Skip to main content
JJOS Demo Journals
HomeJournalsCascadia Journal of Biomedical ResearchVolume 17, Issue 210.58302/dhr02c65
Cascadia Journal of Biomedical Research
Article

JAK-STAT pathway activation compromises nephrocyte function in a Drosophila high-fat diet model of chronic kidney disease: extended analysis

Aiko Watanabe1*, Rosa Bianchi2, Wei Hayashi3

1Department of History, Nordvik University, Norway

2School of Environmental Sciences, University of Cape Highlands, South Africa

3Department of Statistics, Institute for Computational Science, Aurora, Australia

* Corresponding author.

Cascadia J. Biomed. Res. 2025, 17(2); https://doi.org/10.58302/dhr02c65

Abstract

Chronic kidney disease is a major health issue and is gaining prevalence. Using a Drosophila model for chronic kidney disease, we show that a high-fat diet (HFD) disrupts the slit diaphragm filtration structure in nephrocytes, the fly functional equivalent of mammalian podocytes. The structural disruption resulted in reduced filtration function in the affected nephrocytes. We demonstrate that HFD activates the JAK-STAT pathway in nephrocytes, which has previously been linked to diabetic kidney disease. JAK-STAT activation was initiated by increased expression and release of the adipokine, Upd2, from the fat body. This leptin-like hormone is a known ligand of JAK-STAT. Both genetic and pharmacological inhibition of JAK-STAT restored nephrocyte HFD-associated dysfunction. Altogether, our study reveals the importance of the JAK-STAT signaling pathway in the adipose tissue−nephrocyte axis and its contribution to HFD-associated nephropathy. These findings open new avenues for intervention in treating diabetic nephropathy and chronic kidney disease.

Updated in version 2 to clarify the scope of the analysis.

Keywords:JAK-STATDrosophilanephrocyteUpd2Pydslit diaphragmResearch organism D. melanogasterCompellingSolidImportant

Introduction

Chronic kidney disease is a prevalent health issue, with an estimated ~12% of people affected worldwide, many of whom are unaware of their condition (Coresh, 2017; US Department of Health and Human Services and Centers for Disease Control and Prevention (Atlanta, GA), 2023). The gradual loss of kidney function results in excess fluid and the buildup of metabolic waste compounds. Clinical symptoms include atherosclerosis, chronic inflammation, malnutrition, and insulin resistance among other metabolic imbalances (Serrano et al., 2023). Altogether, these lead to increased morbidity and mortality associated with chronic kidney failure (Bikbov et al., 2020). Diabetes, high blood pressure, ageing, obesity, and increased BMI are major risk factors for glomerulopathy and chronic kidney disease (Alizadeh et al., 2019; Berthoux et al., 2013; Bonnet et al., 2001; Coresh, 2017; Hsu et al., 2006; Moorhead et al., 1982; Tsuboi et al., 2013). Notably, patients with primary kidney disease who are also obese have worsened outcomes (Berthoux et al., 2013), and following kidney transplantation, patients with increased BMI are at greater risk of adverse outcomes (Curran et al., 2014), with increasing risk as BMI increases (Hsu et al., 2006). The link between dietary fat intake and kidney disease has raised interest in the adipose-renal axis; that is, how do bodily fat deposits affect kidney function?

Podocytes from patients with chronic kidney disease contain lipid droplets that store excess fat (Herman-Edelstein et al., 2014; Kimmelstiel and Wilson, 1936). These cause lipotoxicity by disrupting the mitochondria, as well as endocytosis (Lubojemska et al., 2021), a process crucial to the kidney filtration structure (Wang et al., 2021). Studies in rats and mice on a HFD have repeatedly shown that the animals suffer from obesity, diabetes (altered insulin homeostasis), and kidney injury marked by functional (albuminuria; blood accumulation of BUN and creatinine; increased urinary biomarkers of kidney damage) and structural (glomerulopathy with glomerular hypertrophy and focal segmental glomerulosclerosis; fibrosis) deficiencies (Altunkaynak et al., 2008; Ha et al., 2022; Jiang et al., 2005; Kuwahara et al., 2016; Lu et al., 2003; Rangel et al., 2019; Ruggiero et al., 2011; Sánchez-Navarro et al., 2021; Sun et al., 2020; Szeto et al., 2016; van der Heijden et al., 2015). Like in patients with chronic kidney disease, lipid droplets have been observed in the kidneys of HFD rats and mice (Deji et al., 2009; Jiang et al., 2005; Sun et al., 2020; van der Heijden et al., 2015) and have been associated with dysfunctional cellular systems, including oxidative stress, renal inflammation, ER stress, disruption of mitochondrial dynamics, and impaired autophagy-lysosomal pathway in the cells of the kidneys (Cai et al., 2024; Ha et al., 2022; Kuwahara et al., 2016; Li et al., 2016b; Lu et al., 2003; Rangel et al., 2019; Ruggiero et al., 2011; Sánchez-Navarro et al., 2021; Sun et al., 2020; Szeto et al., 2016; van der Heijden et al., 2015). Altogether, these indicate the involvement of both local and systemic changes (Deji et al., 2009). However, our understanding of the pathways that govern the pathogenic effect of superfluous fat intake on kidney function, as well as how to leverage this knowledge to develop effective therapeutics, remains incomplete.

Recently, an HFD model in Drosophila showed lipotoxicity, i.e., lipid droplet accumulation, in the fly nephrocytes (Lubojemska et al., 2021). Nephrocytes share many characteristics with human podocytes, including genetics, molecular pathways, and function (Weavers et al., 2009; Zhang et al., 2013a; Zhang et al., 2013b). Both cell types contain highly specialized filtration structures known as slit diaphragms that act in concert with the basement membrane; the fly lacuna channel is similar to the urinary space in the mammalian Bowman’s capsule; and, many key proteins for podocyte function are likewise essential for nephrocyte function (van de Leemput et al., 2022; Wang et al., 2021; Weavers et al., 2009; Zhuang et al., 2009). Indeed, fly in vivo nephrocyte models have been successfully used to study a variety of human kidney diseases, including forms of monogenic nephrotic syndrome and steroid-resistant nephrotic syndrome (SRNS) (Ashraf et al., 2013; Bierzynska et al., 2022; Fu et al., 2017; Gee et al., 2015; Gee et al., 2013; Gonçalves et al., 2018; Hermle et al., 2018; Hermle et al., 2017; Milosavljevic et al., 2022; Odenthal et al., 2023; Paul et al., 2023; Zhao et al., 2019; Zhu et al., 2017). The recent study using the HFD Drosophila model recapitulated the ectopic lipid droplets and cellular dysfunction observed in chronic kidney disease and found that the adipose-derived triglyceride lipase protects nephrocyte endocytosis under HFD conditions (Lubojemska et al., 2021). Here, we used an HFD Drosophila melanogaster model of chronic kidney disease to investigate the role and factors of the adipose-renal axis in chronic kidney disease and identified Upd-activated JAK-STAT signaling as a key component.

Results

HFD compromises nephrocyte function

To investigate the effect of superfluous fat consumption on nephrocyte function, newly eclosed flies were fed a normal fat diet (NFD) or a HFD for 7 days. Then, the flies were subjected to fluorescent dye uptake assays (Weavers et al., 2009; Zhao et al., 2025) to determine pericardial nephrocyte function. HFD leads to enlarged crop (Liao et al., 2021; Zhao et al., 2023a), which was used as an indication of food consistency. Compared to the NFD-fed flies, the HFD-fed flies showed reduced FITC-conjugated albumin (FITC-albumin, 66 kD) fluorescence (Figure 1A and B). Likewise, HFD feeding significantly reduced 10 kD dextran uptake by the nephrocytes (Figure 1C and D). In line with HFD-caused lipid droplet accumulation in the larval nephrocytes (Lubojemska et al., 2021), we observed increased nephrocyte lipid droplets in the adults fed with HFD (Figure 1—figure supplement 1). Thus, confirming the HFD model in Drosophila for studying kidney disease (Lubojemska et al., 2021) and demonstrating that, in our hands, the consumption of superfluous fat caused nephrocyte uptake dysfunction.

High-fat diet compromises nephrocyte function.

Figure 1. 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 leads to lipid droplet accumulation in the nephrocytes.

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).

HFD alters nephrocyte morphology

The slit diaphragm (SD) is the fundamental filtration structure of nephrocytes. Thus, given the HFD-induced dysfunctional uptake, we next tested whether the SD is affected by HFD. Therefore, we looked at the distribution of polychaetoid (Pyd), the fly homolog of human tight junction protein 1 (TJP1, alias ZO-1) and a key component of the SD filtration unit (van de Leemput et al., 2022). Immunostaining with anti-Pyd antibody showed predominant membrane localization of Pyd in nephrocytes from NFD-fed flies. At the cortical surface of NFD nephrocytes, Pyd showed the fingerprint-like pattern characteristic of the SD (Figure 2A). However, in the HFD-fed flies, the SD fingerprint-like pattern appeared irregular and Pyd showed an uneven distribution with spots of increased signal intensity (Figure 2A). These might correlate to the trails of Pyd seemingly hanging from the cortical surface inside the HFD nephrocytes, along with cytosolic accumulation of Pyd protein (Figure 2A and B). Next, we used Sns-mRuby3, in which mRuby3 was tagged at the C-terminal of endogenous Sns (Delaney et al., 2024; Zhao et al., 2025), to verify the HFD effect on SD structure. We observed cytoplasm retention of Sns-mRuby3 in the nephrocytes of HFD-fed flies (Figure 2—figure supplement 1). These findings show that a HFD leads to structural disruption of the SD filtration unit in nephrocytes, that likely accounts for the nephrocyte functional deficit observed earlier.

High-fat diet changes nephrocyte morphology.

Figure 2. 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 changes nephrocyte morphology.

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.

To study the nephrocyte SD and cytoplasmic regions in more detail, we performed transmission electron microscopy (TEM). The nephrocytes of NFD-fed flies showed regularly arranged lacuna channels along the cortical surface (Figure 2C). The distance between the lacuna channels was significantly increased in nephrocytes from HFD-fed flies (Figure 2C and D). Nephrocytes from both NFD and HFD fed flies showed big subcellular vacuoles (Figure 2E). However, the vacuoles in NFD nephrocytes frequently contained electron-dense structures, whereas most vacuoles in HFD nephrocytes were clear (Figure 2E and F). Altogether, these findings demonstrate that HFD causes significant changes to the nephrocyte and its SD, both at the cortical and subcortical levels.

HFD potentiates the JAK-STAT pathway in nephrocytes

Activity of the Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway has been linked to diabetic kidney disease (Berthier et al., 2009); likewise, it is activated systemically in Drosophila in response to a chronic lipid-rich diet (Woodcock et al., 2015). Key components of the JAK-STAT pathway are conserved in flies, including Signal-transducer and activator of transcription protein at 92E (Stat92E), hopscotch (hop), domeless (dome), and Suppressor of cytokine signaling at 36E (Socs36E) (Figure 3A and B). Therefore, we tested whether the JAK-STAT pathway was activated in the nephrocytes of HFD-fed flies. We used a fluorescent reporter 10xStat92E-GFP of JAK-STAT pathway activity (Ekas et al., 2006). Low levels of Stat92E-GFP fluorescence were detected in the nephrocytes of flies on a regular diet (NFD). However, in nephrocytes from HFD-fed flies, the fluorescence was significantly increased (Figure 3C and D). These findings indicate that the consumption of superfluous fat indeed activates the JAK-STAT pathway in nephrocytes.

High-fat diet activates the Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway in nephrocytes.

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.

Activation of the Janus kinase, Hop, reduces nephrocyte function

Since JAK-STAT signaling has been shown to play an important role during development (Brown et al., 2001; Liu et al., 2009), we wanted to isolate its requirement for mature nephrocyte filtration function. To do this, we used a dominant gain-of-function allele of hop (Tumorous-lethal, hop.Tum); a single amino acid change (Gly341Glu) whose expression leads to JAK-STAT pathway activation (Harrison et al., 1995). We expressed UAS-hop.Tum specifically in mature nephrocytes using a temperature-sensitive Dot-Gal4 driver (Dot-Gal4; tub-Gal80ts, referred to as Dot-Gal4ts), known as the TARGET system (McGuire et al., 2004), to turn on expression in adult flies specifically as they are switched to a different environmental temperature (Figure 4A). Overexpression of hop.Tum significantly reduced nephrocyte absorption ability in the adults, as shown with reduced FITC-albumin and 10 kD dextran in Dot-Gal4ts >UAS hop.Tum nephrocytes compared with control nephrocytes (Figure 4B–E).

Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway activation compromises nephrocyte function.

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.

To validate these results and remove between-fly variability, both biological and technical, we also tested the effect of JAK-STAT pathway activation in tissue mosaic clones using Flp-out (Delaney et al., 2024; Duan et al., 2020; Figure 4F). For this technique, first instar larvae are exposed to heat shock to activate the Hsp70P transcription factor, which induces Flippase (Flp). Flp activity, in turn, removes the stop cassette, thereby driving Gal4 expression. Gal4 drives the expression of UAS-GFP and UAS-hop.tum within the same cell. Thus, hop.Tum is only expressed in GFP-labelled nephrocyte clones; whereas, GFP-negative nephrocytes are indicative of no Gal4 being expressed and serve as internal control cells (Figure 4F). In line with the TARGET assays, UAS-hop.Tum overexpression significantly reduced nephrocyte absorption ability in adult flies (Figure 4G and H). These data support a role for the JAK-STAT pathway in nephrocyte function.

Depletion of Socs36E, a negative regulator of JAK-STAT, or increased adipokine Upd2, a JAK-STAT ligand, results in decreased nephrocyte function

Next, we looked at additional JAK-STAT pathway components in the context of nephrocyte function. Suppressor of cytokine signaling at 36E (Socs36E) is transcriptionally regulated by Signal-transducer and activator of transcription 92E (Stat92E) and functions as a negative regulator of JAK-STAT signaling (Callus and Mathey-Prevot, 2002; Karsten et al., 2002; Figure 3B), and it is expressed in the nephrocytes (Figure 3A). Silencing Socs36E specifically in nephrocytes (Dot-Gal4 >Socs36E-RNAi) significantly reduced nephrocyte uptake function as evident in significantly decreased FITC-albumin (Figure 5A and B) and 10 kD dextran (Figure 5C and D) fluorescence compared to the control nephrocytes.

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

Figure 5. 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.

In Drosophila, the JAK-STAT pathway ligand unpaired (Upd) family is encoded by upd1, upd2, and upd3. These ligands bind to Domeless (Dome), a single-transmembrane receptor, to activate JAK-STAT signaling (Figure 3B). Of these, Upd2 is functionally equivalent to human Leptin and is highly expressed in the fat body, the fly’s functional equivalent of vertebrate adipose tissue (Hombría et al., 2005; Rajan et al., 2017; Rajan and Perrimon, 2012). In Drosophila, HFD has been shown to upregulate Upd2 expression and secretion (Rajan et al., 2017; Rajan and Perrimon, 2012). In our model, overexpression of Upd2, specifically in the fat body (ppl-Gal4 driver; ppl-Gal4>upd2 GFP), significantly reduced 10 kD dextran uptake in the nephrocytes compared to the control (ppl-Gal4/+) (Figure 5E and F), indicating reduced nephrocyte function. Since our upd2 over-expression line contains upd2-GFP, we could not use the FITC-albumin uptake assay, as both signals occupy the same imaging channel. Upd2-GFP signal was found in the nephrocytes in ppl-Gal4>upd2 GFP flies (Figure 5—figure supplement 1A), indicating an inter-tissue communication of Upd2-GFP. Notably, Upd2-GFP overexpression in the fat body caused cytoplasm retention of Sns-mRuby3 (Figure 5G and H) and compromised the cortical fingerprint pattern (Figure 5—figure supplement 1B).

Upd2-GFP is secreted from the fat body and transported to the nephrocytes.

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.

Like the Janus kinase Hop, the negative regulator Socs36E and the ligand adipokine Upd2 are additional JAK-STAT pathway components that are required for nephrocyte function.

HFD-induced nephrocyte functional defects are mitigated by Stat92E-mediated inhibition of JAK-STAT

To determine that JAK-STAT forms a direct link between HFD and nephrocyte dysfunction, we knocked down Stat92E (Stat92E-IR; Recasens-Alvarez et al., 2017) in the nephrocytes to disrupt the JAK-STAT signaling pathway (Figure 3B). Deficiency for Stat92E did not affect the uptake function (FITC-albumin or 10 kD dextran) of adult nephrocytes in flies fed a NFD (Figure 6A–D). However, under HFD conditions, in which nephrocytes from control flies showed significantly reduced uptake, Stat92E-deficient nephrocytes showed FITC-albumin and 10 kD dextran fluorescence restored to NFD levels (Figure 6A–D). We used an independent Stat92E-RNAi line in the functional assays and obtained similar results, showing that dextran fluorescence was restored to NFD levels in Stat92E-deficient nephrocytes under HFD conditions (Figure 6—figure supplement 1). Interestingly, under HFD conditions, the cytoplasm retention of Sns-mRuby3 was restored to NFD levels in Stat92E-deficient nephrocytes (Figure 6—figure supplement 2). These findings demonstrate that Stat92E, and by extension the JAK-STAT pathway, is required for the nephrocyte dysfunction induced by HFD.

Silencing Stat92E attenuates nephrocyte functional defects caused by a high-fat diet.

Figure 6. 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).

Stat92E depletion rescues HFD-caused nephrocyte functional decline.

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.

Stat92E depletion rescues HFD-caused Sns-mRuby3 distribution defects in the nephrocytes.

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.

HFD-induced nephrocyte functional defects can be attenuated by methotrexate treatment

Methotrexate suppresses STAT activation; it inhibits the phosphorylation of JAK which is necessary for JAK-STAT pathway activation and was shown to do so without affecting other phosphorylation-dependent pathways (Thomas et al., 2015). In our model system, methotrexate treatment (10 µM; 60 min) reduced levels of Stat92E (10xStat92E-GFP) in the nephrocytes (Figure 7—figure supplement 1). Stat92E is a key component of the pathway; its reduction is indicative of decreased JAK-STAT signaling. Next, we treated flies on NFD or HFD with 10 μM methotrexate (60 min incubation; ex vivo) to study the effect of pharmacological JAK-STAT inhibition on nephrocyte function. In nephrocytes from control flies on a regular diet (NFD), the methotrexate treatment had no effect on uptake of FITC-albumin or 10 kD dextran (Figure 7A–D). However, in flies on a HFD, treatment with methotrexate led to significantly increased nephrocyte FITC-albumin and 10 kD dextran uptake, restoring levels to those observed in control fly NFD nephrocytes (Figure 7A–D). We asked whether methotrexate treatment has an effect on Sns-mRuby3 subcellular distribution. In line with our prediction, under HFD conditions, pharmacological JAK-STAT inhibition restored Sns-mRuby3 distribution to NFD levels (Figure 7—figure supplement 2). Thus, like the genetic intervention (Stat92E inhibition; Figure 6), pharmacological intervention with the JAK-STAT inhibitor methotrexate restored nephrocyte function caused by HFD. These findings support the notion that HFD-induced nephrocyte dysfunction is mediated by the JAK-STAT signaling pathway.

Methotrexate treatment inhibits Janus kinase/signal transducer and activator of transcription (JAK-STA)T pathway activity.

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.

Methotrexate treatment can restore nephrocyte function following a high-fat diet.

Figure 7. 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.

Methotrexate treatment restores Sns-mRuby3 distribution defects following a high-fat diet.

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).

Discussion

A recent publication showed that flies fed a HFD can recapitulate key features of chronic kidney disease, including lipid droplet formation, altered mitochondria dynamics, and endocytosis defects observed as reduced uptake of dextran and albumin (Lubojemska et al., 2021). This previous study found that excess fatty acids, a sign of lipotoxicity, due to a HFD are released from adipose tissue into circulation, then filtered out by the nephrocytes by receptor-mediated endocytosis, at which point the fatty acids accumulate in the lipid droplets (Lubojemska et al., 2021), like those observed in the podocytes of patients with chronic kidney disease (Herman-Edelstein et al., 2014; Kimmelstiel and Wilson, 1936). Notably, lipid droplet lipolysis and PGC1α could counteract the HFD-induced disrupted endocytosis in the nephrocytes via the mitochondria (Lubojemska et al., 2021). This demonstrated one mechanism by which excess lipid droplets, due to HFD, can disrupt nephrocyte kidney function. Here, we likewise used a HFD Drosophila model to study chronic kidney disease and revealed another mechanism by which HFD affects both nephrocyte uptake function and morphology of its filtration structure, the slit diaphragm. HFD upregulates the expression of the adipokine Upd2, the functional homolog to human leptin, which activates the JAK-STAT pathway (Rajan and Perrimon, 2012; Zhao et al., 2023b). Of note, a previous study has shown that Stat1 exerts a repressive effect on PGC1α transcription (Sisler et al., 2015). These observations suggest a potential mechanistic interaction between Jak/Stat signaling and PGC1α regulation. Our data show this also holds true in the nephrocytes, and as such, compromises nephrocyte function (Figure 7E). These findings support obesity as a causal factor in chronic kidney disease.

Of note, in early-stage diabetic kidney disease, JAK-STAT pathway genes are upregulated in patient podocytes (Berthier et al., 2009). In a rat diabetic model, treatment with a JAK-STAT inhibitor (AG-490) reduced proteinuria (Banes et al., 2004); and overexpression of JAK2 in diabetic mouse podocytes elevated JAK-STAT pathway activity and exacerbated diabetic kidney disease (Zhang et al., 2017). These findings, like ours that showed effective treatment of HFD nephropathy using the JAK-STAT inhibitor (methotrexate), support the involvement of the JAK-STAT pathway in HFD-related chronic kidney disease. They also demonstrate that this pathological effect is conserved from flies to mammals. In fact, a phase 2 clinical trial demonstrated that the small molecule baricitinib, a selective JAK1 and JAK2 inhibitor, effectively lowered albuminuria in patients with type 2 diabetes and diabetic kidney disease (Tuttle et al., 2018). Altogether, these studies support JAK-STAT inhibition as a therapeutic intervention for nephropathy associated with superfluous fat intake (Brosius et al., 2016). The conserved disease mechanism makes the HFD fly model a valuable platform to screen JAK-STAT inhibitors for their efficacy to treat chronic kidney disease. The fly findings showed a direct JAK-STAT link at the adipose tissue−nephrocyte axis that leads to nephrocyte dysfunction, via HFD upregulated expression of the adipokine Upd2, which activates the JAK-STAT pathway (Rajan and Perrimon, 2012; Figure 7E). In support, like in our fly model, increased leptin has been observed in obese patients (Considine et al., 1996) and in patients with chronic renal failure (Heimbürger et al., 1997).

Finally, while growing evidence demonstrates that a pharmacological block of the JAK-STAT pathway could effectively treat nephropathy, for decades, the first-line treatment for obesity-related glomerulopathy has been RAS inhibitors (Jiang et al., 2023). Their beneficial effects stem from lowering blood pressure and by lowering the estimated glomerular filtration rate (eGFR) (Banerjee et al., 2022; Yamout et al., 2014). One such inhibitor, telmisartan, targets RAS by blocking the angiotensin (Ang) II receptor and was shown effective in treating nephropathy in a rat model of metabolic syndrome (HFD-fed rats) (Li et al., 2016a). Notably, telmisartan treatment decreased leptin release from adipose tissue, thereby supporting our model and implicating a hitherto unknown mechanism that contributes to leptin-associated nephropathy in metabolic syndrome. In addition, our findings could have implications for lupus nephritis, an inflammatory kidney disease associated with the autoimmune disease systemic lupus erythematosus. Patients with this complication show activated JAK-STAT and elevated leptin, regulated by sterol regulatory element binding transcription factor 1 (SREBF1), which is involved in lipogenesis (Hao et al., 2013). Already, phase 2/3 clinical trials are underway to study the efficacy of JAK inhibitors in treating lupus nephritis (Huo et al., 2023). Our data indicate a possible second pathway could contribute, by direct leptin stimulation of JAK-STAT caused by the dysregulated fat body, warranting further investigation.

Altogether, our study expands our understanding of chronic kidney disease associated with superfluous fat intake and provides new avenues for therapeutic strategies.

Materials and methods

Key resources table

Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
AntibodyChicken polyclonal anti-GFPAbcamCat. ab13970; RRID:AB_300798IF(1:1000)
AntibodyMouse monoclonal anti-PydDevelopmental Studies Hybridoma Bank (DSHB)RRID:AB_2618043IF(1:100)
AntibodyGoat anti-mouse Alexa Fluor 488InvitrogenCat. A11029; RRID:AB_2534088IF(1:500)
AntibodyGoat anti-chicken Alexa Fluor 488InvitrogenCat. A11039; AB_2534096IF(1:500)
Chemical compound, drugMethotrexateSigma-AldrichCas. 06563Methotrexate treatment
OtherDAPIThermo Fisher ScientificCat. D1306Immunochemistry
Other10 kD Texas Red-dextranThermo Fisher ScientificCas. D1828FITC-albumin and 10 kD dextran uptake assays
OtherFITC-albumin solutionSigmaCas. A9771FITC-albumin and 10 kD dextran uptake assays
Genetic reagent (D. melanogaster)Drosophila melanogaster: w1118Bloomington Drosophila Stock Center (BDSC)RRID:BDSC_3605
Genetic reagent (D. melanogaster)Drosophila melanogaster: Dot-Gal4BDSCRRID:BDSC_67608
Genetic reagent (D. melanogaster)Drosophila melanogaster: ppl-Gal4BDSCRRID:BDSC_58768
Genetic reagent (D. melanogaster)Drosophila melanogaster: tub-Gal80tsBDSCRRID:BDSC_7017
Genetic reagent (D. melanogaster)Drosophila melanogaster: 10XStat92E-GFPBDSCRRID:BDSC_26198
Genetic reagent (D. melanogaster)Drosophila melanogaster: Stat92E-IR_#2BDSCRRID:BDSC_33637
Genetic reagent (D. melanogaster)Drosophila melanogaster: Socs36E-IRBDSCRRID:BDSC_35036
Genetic reagent (D. melanogaster)Drosophila melanogaster: Stat92E-IRVienna 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, algorithmFIJI (ImageJ)Schneider et al., 2012; https://imagej.net/Fiji/DownloadsFiji-macOSRRID:SCR_003070
Software, algorithmAdobe Illustrator https://www.adobe.com/ Adobe Illustrator 2022RRID:SCR_010279
Software, algorithmGraphPad Prism https://www.graphpad.com/scientific-software/prism/ GraphPad Prism 9RRID:SCR_002798

Drosophila husbandry

Fly lines were reared on a normal fat diet (NFD; Nutri-Fly German formula; Genesee Scientific, San Diego, CA) or a high-fat diet (HFD; NFD supplemented with 14% coconut oil), under standard conditions (25°C, 60% humidity, 12 hr:12 hr dark:light cycle), unless otherwise stated.

Drosophila stocks w1118 (BDSC_3605), Dot-Gal4 (BDSC_67608), ppl-Gal4 (BDSC_58768), tub-Gal80ts (BDSC_7017), 10XStat92E-GFP (BDSC_26198), UAS-Stat92E-RNAi_#2 (BDSC_33637), and UAS-Socs36E-RNAi (BDSC_35036) were obtained from the Bloomington Drosophila Stock Center (BDSC). UAS-Stat92E-RNAi (VDRC_106980) was obtained from the Vienna Drosophila Resource Center (VDRC). UAS-hop.Tum was kindly provided by Prof. Norbert Perrimon (Harvard Medical School, Boston, MA; Howard Hughes Medical Institute, Boston, MA). UAS-upd2:GFP has been previously described (Hombría et al., 2005) and was kindly provided by Prof. Ylva Engström (Stockholm University, Stockholm, Sweden). The Flp-out line hs-Flp122; UAS-FlpJD1/CyO, Act-GFPJMR1; Act5C>CD2>Gal4S, UAS-mCD8-GFPLL6/TM6b was generated previously (Zhao et al., 2015).

FITC-albumin and 10 kD dextran uptake assays

Nephrocyte functional assays were performed ex vivo at room temperature, following a previously described method (Wen et al., 2020) with minor changes. Drosophila females were dissected in Schneider’s Drosophila Medium (Thermo Fisher Scientific, MA), then incubated in a 10 kD Texas Red-dextran solution (0.05 mg/mL; D1828, Thermo Fisher Scientific, MA) in Schneider’s Drosophila Medium (Thermo Fisher Scientific, MA) for 20 min, or a FITC-albumin solution (10 mM; Sigma, A9771) in Schneider’s Drosophila Medium (Thermo Fisher Scientific, MA) for 5 min. The specimens were washed in artificial hemolymph twice, followed by fixation in 4% paraformaldehyde (PFA) for 60 min. Then the fixed specimens were washed thrice for 5 min in 1x phosphate buffered saline (1xPBS; pH 7.4) and mounted using Vectashield mounting medium (H-1000, Vector Laboratories, CA). FITC-albumin and 10 kD dextran specimens were imaged using a ZEISS LSM900 confocal microscope with ZEISS Zen acquisition software (blue edition; version 3.0) using a 20× Plan-Apochromat 0.8 N.A. air objective (ZEISS, Oberkochen, Germany). For quantitative comparison of fluorescence intensities, settings for the control condition were chosen to avoid oversaturation (using Range Indicator in ZEN blue; limiting the observed red dots to avoid oversaturation), then applied across the images for all samples/conditions within the assay. The fluorescence intensity of FITC-albumin and 10 kD dextran nephrocytes was determined using Fiji software (Image J; Schneider et al., 2012, version 2.9.0/1.53t; National Institutes of Health, Bethesda). For quantitation, for each genotype, the relative fluorescence intensity of 30 nephrocytes from 6 female flies (five nephrocytes/fly) was analyzed.

Immunochemistry

Immunostaining was performed as previously reported (Zhao et al., 2022) with minor changes. Flies were briefly rinsed in 95% ethanol and dissected in 1xPBS at room temperature. Specimens were incubated in primary antibodies overnight at 4°C. The incubations in secondary antibodies were performed either overnight at 4°C or for 2 hr at room temperature. The following antibodies were used: chicken anti-GFP (1:1,000; ab13970, RRID:AB_300798, Abcam, Cambridge, UK), mouse monoclonal anti-Pyd (1:100; RRID:AB_2618043, Developmental Studies Hybridoma Bank, IA), goat anti-mouse Alexa Fluor 488 (1:500; A11029, RRID:AB_2534088, Invitrogen, Eugene, OR), and goat anti-chicken Alexa Fluor 488 (1:500; A11039, AB_2534096, Invitrogen, Eugene, OR). DAPI (0.5 mg/ml in PBST (0.2% Triton X-100 in 1x PBS); D1306, Thermo Fisher Scientific, MA) was used to visualize the nuclei. The nephrocytes were imaged using a ZEISS LSM900 confocal microscope (under Airyscan mode for Pyd images) with ZEISS ZEN acquisition software (blue edition; version 3.0) and a 63x Plan-Apochromat 1.4 N.A. oil objective (ZEISS, Oberkochen, Germany). For quantitative comparison of fluorescence intensities, settings for the control condition were chosen to avoid oversaturation (using Range Indicator in ZEN blue; limiting the observed red dots to avoid oversaturation), then applied across the images for all samples/conditions within the assay. Image J (Schneider et al., 2012) was used for image processing (version 2.9.0/1.53t; National Institutes of Health, Bethesda, MD). Typically, six flies were imaged per condition, and representative images for each are displayed in the figures.

Transmission electron microscopy (TEM)

TEM was performed using standardized procedures. In brief, female adults (7 days old) were dissected in Schneider’s Drosophila Medium (Thermo Fisher Scientific, MA). The heart tube and the attached nephrocytes were dissected, removed, and fixed using Sorensen phosphate buffer (2% PFA, 2.5% EM grade glutaraldehyde, 2 mM CaCl2, 0.1 M NaOH; provided by the Electron Microscopy Core Imaging Facility at the Center for Innovative Biomedical Resources (CIBR), University of Maryland School of Medicine, MD). The processed specimens were imaged using a Philips CM100 TEM, carried out at the Electron Microscopy Core Imaging Facility at the Center for Innovative Biomedical Resources (CIBR) (University of Maryland School of Medicine, MD). The LC-LC distances were measured using the Straight Line tool in Fiji (Image J Schneider et al., 2012, version 2.9.0/1.53t; National Institutes of Health, Bethesda, MD) to connect two adjacent lacuna channels (LCs); a straight line was drawn from the middle of the first LC to the middle of the sixth LC, covering five LC-LC intervals on a TEM image, then the measure function was used to obtain the distance value. In total, 60 LC-LC distances were measured per condition in 10 nephrocytes from six 7-day-old female flies. The presence of a dark electron-dense structure in the vacuoles was manually determined and counted in images obtained from 12 nephrocytes for NFD and 29 nephrocytes from HFD from six 7-day-old female flies.

Tissue mosaic analysis

Flp-out clone (Struhl and Basler, 1993) induction was performed as previously described (Duan et al., 2020). In brief, female virgins of hs-Flp122; UAS-FlpJD1/CyO, Act-GFPJMR1; Act5C>CD2>Gal4S, UAS-mCD8-GFPLL6/TM6b (Zhao et al., 2015) were crossed with UAS-hop.Tum males. The embryos were collected for 24 hr. First instar larvae (24 hr after embryo collection) received a 10 min heat shock in a 37°C water bath to induce the mosaic clones. After the heat shock, the larvae were maintained at 25°C. One-day-old female adults were subjected to the 10 kD dextran functional assay (described above). The GFP-positive nephrocyte clones and their neighboring nephrocytes were analyzed.

Methotrexate treatment

Methotrexate (06563, Sigma-Aldrich, MO) was dissolved in DMSO to make a 10 mM stock solution. Flies were dissected as described for the nephrocyte functional assays. The dorsal cuticle (with nephrocytes) was transferred to methotrexate solution (10 μM) in Schneider’s Drosophila Medium (Thermo Fisher Scientific, MA) and incubated at room temperature for 60 min. The samples incubated in Schneider’s Medium supplemented with DMSO vehicle were used as a control. The specimens were rinsed with Schneider’s Drosophila Medium (Thermo Fisher Scientific, MA), then subjected to a functional assay or fixed in 4% PFA for immunochemistry.

Nie Red staining

Newly eclosed w1118 flies were fed either a high-fat diet (HFD) or a control diet (CD) for 7 days before staining for lipid droplets using Nile Red. For Nile Red staining, nephrocytes were dissected in 1× PBS and fixed in 4% paraformaldehyde for 1 hr at room temperature. The samples were then washed three times with 1× PBS for 5 min each, followed by incubation with 0.1 μg/mL Nile Red (HY-D0718, MedChemExpress) for 10 min at room temperature. Afterward, the samples were rinsed three more times with 1x PBS for 5 min each.

Data analyses and figure preparation

Image J (Schneider et al., 2012) (version .9.0/1.53t; National Institutes of Health, Bethesda, MD) was used to process the raw data of confocal images and to measure the relative fluorescence intensity. The data sets were tested for normality using the Shapiro-Wilk test and plotted using GraphPad Prism 9 software (version 9.5.1). Normally distributed data were analyzed by the two-tailed Student’s t-test, or by two-way ANOVA with Sidak correction. P<0.05 was considered significant. The figures were arranged using Adobe Illustrator software (version 2022 26.2.1). Box plots show the median (center line), interquartile range (25th–75th percentiles; box), and whiskers extending to the minimum and maximum values.

Funding Information

This paper was supported by the following grants:

  • https://ror.org/00adh9b73National Institute of Diabetes and Digestive and Kidney Diseases R01-DK098410 to Zhe Han.

  • https://ror.org/00adh9b73National Institute of Diabetes and Digestive and Kidney Diseases R01-DK140937 to Zhe Han.

Acknowledgements

We thank the Bloomington Drosophila Stock Center (BDSC) based at Indiana University (Bloomington, IN), the Vienna Drosophila Resource Center (VDRC) based at Vienna BioCenter (Vienna, Austria), Prof. Norbert Perrimon (Harvard Medical School, Boston, MA; Howard Hughes Medical Institute, Boston, MA), and Prof. Ylva Engström (Stockholm University, Stockholm, Sweden) for sharing Drosophila stocks; and the Developmental Studies Hybridoma Bank (DSHB) based at the University of Iowa (Iowa City, IA) for providing the antibodies. Finally, we thank the Electron Microscopy Core Imaging Facility at the Center for Innovative Biomedical Resources (CIBR) (University of Maryland School of Medicine, MD, USA) for their support in TEM image acquisition.

Additional information

Competing interests

No competing interests declared.

Author contributions

Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review and editing.

Conceptualization, Investigation, Methodology, Writing – original draft.

Investigation.

Writing – original draft, Writing – review and editing.

Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Writing – original draft, Project administration, Writing – review and editing.

Additional files

MDAR checklist
Source data 1. Individual values for each condition and genotype shown in the plots.

References

  1. AlizadehS EsmaeiliH AlizadehM DaneshzadE SharifiL RadfarH RadaeiMK 2019Metabolic phenotypes of obese, overweight, and normal weight individuals and risk of chronic kidney disease: a systematic review and meta-analysisArchives of Endocrinology and Metabolism6342743710.20945/2359-399700000014931365625PMC10528657
  2. AltunkaynakME OzbekE AltunkaynakBZ CanI UnalD UnalB 2008The effects of high-fat diet on the renal structure and morphometric parametric of kidneys in ratsJournal of Anatomy21284585210.1111/j.1469-7580.2008.00902.x18510511PMC2423405
  3. AshrafS GeeHY WoernerS XieLX Vega-WarnerV LovricS FangH SongX CattranDC Avila-CasadoC PatersonAD NitschkéP Bole-FeysotC CochatP Esteve-RuddJ HaberbergerB AllenSJ ZhouW AirikR OttoEA BaruaM Al-HamedMH KariJA EvansJ BierzynskaA SaleemMA BöckenhauerD KletaR El DesokyS HacihamdiogluDO GokF WashburnJ WigginsRC ChoiM LiftonRP LevyS HanZ SalviatiL ProkischH WilliamsDS PollakM ClarkeCF PeiY AntignacC HildebrandtF 2013ADCK4 mutations promote steroid-resistant nephrotic syndrome through CoQ10 biosynthesis disruptionThe Journal of Clinical Investigation1235179518910.1172/JCI6900024270420PMC3859425
  4. BanerjeeD WinocourP ChowdhuryTA DeP WahbaM MonteroR FogartyD FrankelAH KarallieddeJ MarkPB PatelDC PokrajacA SharifA Zac-VargheseS BainS DasguptaI 2022Association of British clinical diabetologists and the renal associationBMC Nephrology23910.1186/s12882-021-02587-534979961PMC8722287
  5. BanesAK ShawS JenkinsJ ReddH AmiriF PollockDM MarreroMB 2004Angiotensin II blockade prevents hyperglycemia-induced activation of JAK and STAT proteins in diabetic rat kidney glomeruliAmerican Journal of Physiology. Renal Physiology286F653F65910.1152/ajprenal.00163.200314678947
  6. BerthierCC ZhangH SchinM HengerA NelsonRG YeeB BoucherotA NeusserMA CohenCD Carter-SuC ArgetsingerLS RastaldiMP BrosiusFC KretzlerM 2009Enhanced expression of Janus kinase-signal transducer and activator of transcription pathway members in human diabetic nephropathyDiabetes5846947710.2337/db08-132819017763PMC2628622
  7. BerthouxF MariatC MaillardN 2013Overweight/obesity revisited as a predictive risk factor in primary IgA nephropathyNephrology Dialysis Transplantation28iv160iv16610.1093/ndt/gft28624026246
  8. BierzynskaA BullK MielletS DeanP NealC ColbyE McCarthyHJ HegdeS SinhaMD Bugarin DizC StirrupsK MegyK MapetaR PenkettC MarshS ForresterN AfzalM StarkH BioResourceN WilliamsM WelshGI KoziellAB HartleyPS SaleemMA 2022Exploring the relevance of NUP93 variants in steroid-resistant nephrotic syndrome using next generation sequencing and a fly kidney modelPediatric Nephrology372643265610.1007/s00467-022-05440-535211795PMC9489583
  9. BikbovB PurcellCA LeveyAS SmithM AbdoliA AbebeM AdebayoOM AfaridehM AgarwalSK Agudelo-BoteroM AhmadianE Al-AlyZ AlipourV Almasi-HashianiA Al-RaddadiRM Alvis-GuzmanN AminiS AndreiT AndreiCL AndualemZ AnjomshoaM ArablooJ AshagreAF AsmelashD AtaroZ AtoutMMW AyanoreMA BadawiA BakhtiariA BallewSH BalouchiA BanachM BarqueraS BasuS BayihMT BediN BelloAK BensenorIM BijaniA BoloorA BorzìAM CámeraLA CarreroJJ CarvalhoF CastroF Catalá-LópezF ChangAR ChinKL ChungSC CirilloM CousinE DandonaL DandonaR DaryaniA Das GuptaR DemekeFM DemozGT DestaDM DoHP DuncanBB EftekhariA EsteghamatiA FatimaSS FernandesJC FernandesE FischerF FreitasM GadMM GebremeskelGG GebresillassieBM GetaB GhafourifardM GhajarA GhithN GillPS GinawiIA GuptaR Hafezi-NejadN Haj-MirzaianA Haj-MirzaianA HariyaniN HasanM HasankhaniM HasanzadehA HassenHY HaySI HeidariB HerteliuC HoangCL HosseiniM HostiucM IrvaniSSN IslamSMS Jafari BalalamiN JamesSL JassalSK JhaV JonasJB JoukarF JozwiakJJ KabirA KahsayA KasaeianA KassaTD KassayeHG KhaderYS KhalilovR KhanEA KhanMS KhangYH KisaA KovesdyCP Kuate DefoB KumarGA LarssonAO LimLL LopezAD LotufoPA MajeedA MalekzadehR MärzW MasakaA MeheretuHAA MiazgowskiT MiricaA MirrakhimovEM MithraP MoazenB MohammadDK MohammadpourhodkiR MohammedS MokdadAH MoralesL Moreno VelasquezI MousaviSM MukhopadhyayS NachegaJB NadkarniGN NansseuJR NatarajanG NazariJ NealB NegoiRI NguyenCT NikbakhshR NoubiapJJ NowakC OlagunjuAT OrtizA OwolabiMO PalladinoR PathakM PoustchiH PrakashS PrasadN RafieiA RajuSB RamezanzadehK RawafS RawafDL RawalL ReinerRC RezapourA RibeiroDC RoeverL RothenbacherD RwegereraGM SaadatagahS SafariS SahleBW SalemH SanabriaJ SantosIS SarveazadA SawhneyM SchaeffnerE SchmidtMI SchutteAE SepanlouSG ShaikhMA SharafiZ SharifM SharifiA SilvaDAS SinghJA SinghNP SisayMMM SoheiliA SutradharI TeklehaimanotBF TesfayBE TeshomeGF ThakurJS TonelliM TranKB TranBX Tran NgocC UllahI ValdezPR VarugheseS VosT VuLG WaheedY WerdeckerA WoldeHF WondmienehAB Wulf HansonS YamadaT YeshawY YonemotoN YusefzadehH ZaidiZ ZakiL ZamanSB ZamoraN ZarghiA ZewdieKA ÄrnlövJ CoreshJ PericoN RemuzziG MurrayCJL VosT 2020Global, regional, and national burden of chronic kidney disease, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017The Lancet39570973310.1016/S0140-6736(20)30045-3PMC704990532061315
  10. BonnetF DepreleC SassolasA MoulinP AlamartineE BerthezèneF BerthouxF 2001Excessive body weight as a new independent risk factor for clinical and pathological progression in primary IgA nephritisAmerican Journal of Kidney Diseases3772072710.1016/s0272-6386(01)80120-711273871
  11. BrosiusFC TuttleKR KretzlerM 2016JAK inhibition in the treatment of diabetic kidney diseaseDiabetologia591624162710.1007/s00125-016-4021-527333885PMC4942738
  12. BrownS HuN HombríaJC 2001Identification of the first invertebrate interleukin JAK/STAT receptor, the Drosophila gene domelessCurrent Biology111700170510.1016/s0960-9822(01)00524-311696329
  13. CaiMY JiangXS WeiYX WenRZ DuXG 2024Role of TFEB-autophagy lysosomal pathway in palmitic acid induced renal tubular epithelial cell injuryBiochemical and Biophysical Research Communications6961494723824180910.1016/j.bbrc.2024.149472
  14. CallusBA Mathey-PrevotB 2002SOCS36E, a novel Drosophila SOCS protein, suppresses JAK/STAT and EGF-R signalling in the imaginal wing discOncogene214812482110.1038/sj.onc.120561812101419
  15. ConsidineRV SinhaMK HeimanML KriauciunasA StephensTW NyceMR OhannesianJP MarcoCC McKeeLJ BauerTL 1996Serum immunoreactive-leptin concentrations in normal-weight and obese humansThe New England Journal of Medicine33429229510.1056/NEJM1996020133405038532024
  16. CoreshJ 2017Update on the burden of CKDJournal of the American Society of Nephrology281020102210.1681/ASN.201612137428302756PMC5373470
  17. CurranSP FamureO LiY KimSJ 2014Increased recipient body mass index is associated with acute rejection and other adverse outcomes after kidney transplantationTransplantation97647010.1097/TP.0b013e3182a688a424056619
  18. DejiN KumeS ArakiSI SoumuraM SugimotoT IsshikiK Chin-KanasakiM SakaguchiM KoyaD HanedaM KashiwagiA UzuT 2009Structural and functional changes in the kidneys of high-fat diet-induced obese miceAmerican Journal of Physiology-Renal Physiology296F118F12610.1152/ajprenal.00110.200818971213
  19. DelaneyM ZhaoY van de LeemputJ LeeH HanZ 2024Actin cytoskeleton and integrin components are interdependent for slit diaphragm maintenance in Drosophila NephrocytesCells13135010.3390/cells1316135039195240PMC11352372
  20. DuanJ ZhaoY LiH HabernigL GordonMD MiaoX EngströmY BüttnerS 2020Bab2 functions as an ecdysone-responsive transcriptional repressor during Drosophila developmentCell Reports3210797210.1016/j.celrep.2020.10797232726635
  21. EkasLA BaegGH FlahertyMS Ayala-CamargoA BachEA 2006JAK/STAT signaling promotes regional specification by negatively regulating wingless expression in DrosophilaDevelopment1334721472910.1242/dev.0267517079268
  22. FuY ZhuJY RichmanA ZhaoZ ZhangF RayPE HanZ 2017A Drosophila model system to assess the function of human monogenic podocyte mutations that cause nephrotic syndromeHuman Molecular Genetics2676878010.1093/hmg/ddw42828164240PMC6074792
  23. GeeHY SaisawatP AshrafS HurdTW Vega-WarnerV FangH BeckBB GribouvalO ZhouW DiazKA NatarajanS WigginsRC LovricS CherninG SchoebDS OvuncB FrishbergY SolimanNA FathyHM GoebelH HoefeleJ WeberLT InnisJW FaulC HanZ WashburnJ AntignacC LevyS OttoEA HildebrandtF 2013ARHGDIA mutations cause nephrotic syndrome via defective RHO GTPase signalingThe Journal of Clinical Investigation1233243325310.1172/JCI6913423867502PMC3726174
  24. GeeHY ZhangF AshrafS KohlS SadowskiCE Vega-WarnerV ZhouW LovricS FangH NettletonM ZhuJ HoefeleJ WeberLT PodrackaL BoorA FehrenbachH InnisJW WashburnJ LevyS LiftonRP OttoEA HanZ HildebrandtF 2015KANK deficiency leads to podocyte dysfunction and nephrotic syndromeThe Journal of Clinical Investigation1252375238410.1172/JCI7950425961457PMC4497755
  25. GonçalvesS PatatJ GuidaMC LachausséeN ArrondelC HelmstädterM BoyerO GribouvalO GublerMC MolletG RioM CharbitM Bole-FeysotC NitschkeP HuberTB WheelerPG HaynesD JuusolaJ Billette de VillemeurT NavaC AfenjarA KerenB BodmerR AntignacC SimonsM 2018A homozygous KAT2B variant modulates the clinical phenotype of ADD3 deficiency in humans and fliesPLOS Genetics14e100738610.1371/journal.pgen.100738629768408PMC5973622
  26. HaS YangY KimBM KimJ SonM KimD YuHS ImD ChungHY ChungKW 2022Activation of PAR2 promotes high-fat diet-induced renal injury by inducing oxidative stress and inflammationBiochimica et Biophysica Acta - Molecular Basis of Disease186816647410.1016/j.bbadis.2022.16647435772632
  27. HaoJ ZhangY LvX XuN LiuQ ZhaoS FengX XingL KangP LiG LiuS 2013IFN-γ induces lipogenesis in mouse mesangial cells via the JAK2/STAT1 pathwayAmerican Journal of Physiology-Cell Physiology304C760C76710.1152/ajpcell.00352.201223407880
  28. HarrisonDA BinariR NahreiniTS GilmanM PerrimonN 1995Activation of a Drosophila Janus kinase (JAK) causes hematopoietic neoplasia and developmental defectsThe EMBO Journal142857286510.1002/j.1460-2075.1995.tb07285.x7796812PMC398404
  29. HeimbürgerO LönnqvistF DanielssonA NordenströmJ StenvinkelP 1997Serum immunoreactive leptin concentration and its relation to the body fat content in chronic renal failureJournal of the American Society of Nephrology81423143010.1681/ASN.V8914239294834
  30. Herman-EdelsteinM ScherzerP TobarA LeviM GafterU 2014Altered renal lipid metabolism and renal lipid accumulation in human diabetic nephropathyJournal of Lipid Research5556157210.1194/jlr.P04050124371263PMC3934740
  31. HermleT BraunDA HelmstädterM HuberTB HildebrandtF 2017Modeling monogenic human nephrotic syndrome in the drosophila garland cell nephrocyteJournal of the American Society of Nephrology: JASN281521153310.1681/ASN.201605051727932481PMC5407722
  32. HermleT SchneiderR SchapiroD BraunDA van der VenAT WarejkoJK DagaA WidmeierE NakayamaM Jobst-SchwanT MajmundarAJ AshrafS RaoJ FinnLS TasicV HernandezJD BaggaA JalalahSM El DesokyS KariJA LaricchiaKM LekM RehmHL MacArthurDG ManeS LiftonRP ShrilS HildebrandtF 2018GAPVD1 and ANKFY1 mutations implicate RAB5 regulation in nephrotic syndromeJournal of the American Society of Nephrology292123213810.1681/ASN.201712131229959197PMC6065084
  33. HombríaJCG BrownS HäderS ZeidlerMP 2005Characterisation of Upd2, a Drosophila JAK/STAT pathway ligandDevelopmental Biology28842043310.1016/j.ydbio.2005.09.04016277982
  34. HsuC McCullochCE IribarrenC DarbinianJ GoAS 2006Body mass index and risk for end-stage renal diseaseAnnals of Internal Medicine144212810.7326/0003-4819-144-1-200601030-0000616389251
  35. HuoR HuangX YangY LinJ 2023Potential use of janus kinase inhibitors in the treatment of systemic lupus erythematosusJournal of Inflammation Research161471147810.2147/JIR.S39763937051062PMC10084827
  36. JiangT WangZ ProctorG MoskowitzS LiebmanSE RogersT LuciaMS LiJ LeviM 2005Diet-induced obesity in C57BL/6J mice causes increased renal lipid accumulation and glomerulosclerosis via a sterol regulatory element-binding protein-1c-dependent pathwayThe Journal of Biological Chemistry280323173232510.1074/jbc.M50080120016046411
  37. JiangZ WangY ZhaoX CuiH HanM RenX GangX WangG 2023Obesity and chronic kidney diseaseAmerican Journal of Physiology. Endocrinology and Metabolism324E24E4110.1152/ajpendo.00179.202236383637
  38. KarstenP HäderS ZeidlerMP 2002Cloning and expression of Drosophila SOCS36E and its potential regulation by the JAK/STAT pathwayMechanisms of Development11734334610.1016/s0925-4773(02)00216-212204282
  39. KimmelstielP WilsonC 1936Intercapillary lesions in the glomeruli of the kidneyThe American Journal of Pathology12839819970254PMC1911022
  40. KuwaharaS HosojimaM KanekoR AokiH NakanoD SasagawaT KabasawaH KasedaR YasukawaR IshikawaT SuzukiA SatoH KageyamaS TanakaT KitamuraN NaritaI KomatsuM NishiyamaA SaitoA 2016Megalin-mediated tubuloglomerular alterations in high-fat diet-induced kidney diseaseJournal of the American Society of Nephrology271996200810.1681/ASN.201502019026534923PMC4926965
  41. LiH LiM LiuP WangY ZhangH LiH YangS SongY YinY GaoL ChengS CaiJ TianG 2016aTelmisartan ameliorates nephropathy in metabolic syndrome by reducing leptin release from perirenal adipose tissueHypertension6847849010.1161/HYPERTENSIONAHA.116.0700827296996
  42. LiC LinY LuoR ChenS WangF ZhengP LeviM YangT WangW 2016bIntrarenal renin-angiotensin system mediates fatty acid-induced ER stress in the kidneyAmerican Journal of Physiology. Renal Physiology310F351F36310.1152/ajprenal.00223.201526672616PMC4971807
  43. LiaoS AmcoffM NässelDR 2021Impact of high-fat diet on lifespan, metabolism, fecundity and behavioral senescence in DrosophilaInsect Biochemistry and Molecular Biology13310349510.1016/j.ibmb.2020.10349533171202
  44. LiuYH JakobsenJS ValentinG AmarantosI GilmourDT FurlongEEM 2009A systematic analysis of Tinman function reveals Eya and JAK-STAT signaling as essential regulators of muscle developmentDevelopmental Cell1628029110.1016/j.devcel.2009.01.00619217429
  45. LuJ Bankovic-CalicN OgbornM SaboorianMH AukemaHM 2003Detrimental effects of a high fat diet in early renal injury are ameliorated by fish oil in Han:SPRD-cy ratsThe Journal of Nutrition13318018610.1093/jn/133.1.18012514287
  46. LubojemskaA StefanaMI SorgeS BaileyAP LampeL YoshimuraA BurrellA CollinsonL GouldAP 2021Adipose triglyceride lipase protects renal cell endocytosis in a Drosophila dietary model of chronic kidney diseasePLOS Biology19e300123010.1371/journal.pbio.300123033945525PMC8121332
  47. McGuireSE MaoZ DavisRL 2004Spatiotemporal gene expression targeting with the TARGET and gene-switch systems in DrosophilaScience’s STKE20041610.1126/stke.2202004pl614970377
  48. MilosavljevicJ LempickiC LangK HeinkeleH KampfLL LeroyC ChenM GerstnerL SpitzD WangM KnobAU KayserS HelmstädterM WalzG PollakMR HermleT 2022Nephrotic syndrome gene TBC1D8B is required for endosomal maturation and nephrin endocytosis in DrosophilaJournal of the American Society of Nephrology332174219310.1681/ASN.202203027536137753PMC9731638
  49. MoorheadJF El-NahasM ChanMK VargheseZ 1982Lipid nephrotoxicity in chronic progressive glomerular and tubulo-interstitial diseaseLancet3201309131110.1016/S0140-6736(82)91513-66128601
  50. OdenthalJ DittrichS LudwigV MerzT ReitmeierK ReuschB HöhneM CosgunZC HohenadelM PutnikJ GöbelH RinschenMM AltmüllerJ KoehlerS SchermerB BenzingT BeckBB BrinkkötterPT HabbigS BartramMP 2023Modeling of ACTN4-based podocytopathy using Drosophila nephrocytesKidney International Reports831732910.1016/j.ekir.2022.10.02436815115PMC9939316
  51. PaulMS DuncanAR GenettiCA PanH JacksonA GrantPE ShiJ PinelliM Brunetti-PierriN Garza-FloresA ShahaniD SanetoRP ZampinoG LeoniC AgoliniE NovelliA BlümleinU HaackTB HeinritzW MatzkerE AlhaddadB Abou JamraR BartolomaeusT AlHamdanS CarapitoR IsidorB BahramS RitterA IzumiK ShakkedBP BarelO Ben ZeevB BegtrupA CarereDA MullegamaSV PalculictTB CalameDG SchwanK AycinenaARP TrabergR ConsortiumGER DouzgouS PirtH IsmayilovaN BankaS ChaoHT AgrawalPB 2023Rare EIF4A2 variants are associated with a neurodevelopmental disorder characterized by intellectual disability, hypotonia, and epilepsyAmerican Journal of Human Genetics11012014510.1016/j.ajhg.2022.11.01136528028PMC9892767
  52. RajanA PerrimonN 2012Drosophila cytokine unpaired 2 regulates physiological homeostasis by remotely controlling insulin secretionCell15112313710.1016/j.cell.2012.08.01923021220PMC3475207
  53. RajanA HousdenBE Wirtz-PeitzF HolderbaumL PerrimonN RajanA HousdenBE Wirtz-PeitzF HolderbaumL PerrimonN RajanA HousdenBE Wirtz-PeitzF HolderbaumL PerrimonN RajanA HousdenBE Wirtz-PeitzF HolderbaumL PerrimonN RajanA HousdenBE Wirtz-PeitzF HolderbaumL PerrimonN RajanA HousdenBE Wirtz-PeitzF HolderbaumL PerrimonN 2017A mechanism coupling systemic energy sensing to adipokine secretionDevelopmental Cell43839810.1016/j.devcel.2017.09.00729017032PMC5650192
  54. RangelSR Nunes Goulart da Silva PereiraE Eduardo Ilaquita FloresE Lino RodriguesK Ribeiro SilvaA Gonçalves-de-AlbuquerqueCF DaliryA 2019High-fat diet-induced kidney alterations in rats with metabolic syndrome: endothelial dysfunction and decreased antioxidant defenseDiabetes, Metabolic Syndrome and Obesity121773178110.2147/DMSO.S21125331564943PMC6735540
  55. Recasens-AlvarezC FerreiraA MilánM 2017JAK/STAT controls organ size and fate specification by regulating morphogen production and signallingNature Communications81381510.1038/ncomms1381528045022PMC5216089
  56. RuggieroC EhrenshaftM ClelandE StadlerK 2011High-fat diet induces an initial adaptation of mitochondrial bioenergetics in the kidney despite evident oxidative stress and mitochondrial ROS productionAmerican Journal of Physiology. Endocrinology and Metabolism300E1047E105810.1152/ajpendo.00666.201021386058PMC3118596
  57. Sánchez-NavarroA Martínez-RojasMÁ Caldiño-BohnRI Pérez-VillalvaR ZambranoE Castro-RodríguezDC BobadillaNA 2021Early triggers of moderately high-fat diet-induced kidney damagePhysiological Reports9e1493710.14814/phy2.1493734291592PMC8295594
  58. SchneiderCA RasbandWS EliceiriKW 2012NIH Image to ImageJ: 25 years of image analysisNature Methods967167510.1038/nmeth.208922930834PMC5554542
  59. SerranoE ShenoyP Martinez CantarinMP 2023Adipose tissue metabolic changes in chronic kidney diseaseImmunometabolism5e0002310.1097/IN9.000000000000002337128293PMC10144329
  60. SislerJD MorganM RajeV GrandeRC DereckaM MeierJ CantwellM SzczepanekK KorzunWJ LesnefskyEJ HarrisTE CronigerCM LarnerAC 2015The Signal Transducer and Activator of Transcription 1 (STAT1) inhibits mitochondrial biogenesis in liver and fatty acid oxidation in adipocytesPLOS ONE10e014444410.1371/journal.pone.014444426689548PMC4686975
  61. StruhlG BaslerK 1993Organizing activity of wingless protein in DrosophilaCell7252754010.1016/0092-8674(93)90072-x8440019
  62. SunY GeX LiX HeJ WeiX DuJ SunJ LiX XunZ LiuW ZhangH WangZY LiYC 2020High-fat diet promotes renal injury by inducing oxidative stress and mitochondrial dysfunctionCell Death & Disease1111410.1038/s41419-020-03122-433099578PMC7585574
  63. SzetoHH LiuS SoongY AlamN PruskyGT SeshanSV 2016Protection of mitochondria prevents high-fat diet-induced glomerulopathy and proximal tubular injuryKidney International90997101110.1016/j.kint.2016.06.01327519664
  64. ThomasS FisherKH SnowdenJA DansonSJ BrownS ZeidlerMP 2015Methotrexate Is a JAK/STAT pathway inhibitorPLOS ONE10e013007810.1371/journal.pone.013007826131691PMC4489434
  65. TsuboiN KoikeK HiranoK UtsunomiyaY KawamuraT HosoyaT 2013Clinical features and long-term renal outcomes of Japanese patients with obesity-related glomerulopathyClinical and Experimental Nephrology1737938510.1007/s10157-012-0719-y23135866
  66. TuttleKR BrosiusFCIII AdlerSG KretzlerM MehtaRL TumlinJA TanakaY HanedaM LiuJ SilkME CardilloTE DuffinKL HaasJV MaciasWL NunesFP JanesJM 2018JAK1/JAK2 inhibition by baricitinib in diabetic kidney disease: results from a Phase 2 randomized controlled clinical trialNephrology Dialysis Transplantation331950195910.1093/ndt/gfx377PMC621272029481660
  67. US Department of Health and Human Services Centers for Disease Control and Prevention (Atlanta, GA) 2023Chronic Kidney Disease in the United Stateshttps://www.cdc.gov/kidneydisease/publications-resources/ckd-national-facts.htmlJune 1, 2023
  68. van de LeemputJ WenP HanZ 2022Using Drosophila Nephrocytes to understand the formation and maintenance of the podocyte slit diaphragmFrontiers in Cell and Developmental Biology1083782810.3389/fcell.2022.83782835265622PMC8898902
  69. van der HeijdenRA BijzetJ MeijersWC YakalaGK KleemannR NguyenTQ de BoerRA SchalkwijkCG HazenbergBPC TietgeUJF HeeringaP 2015Obesity-induced chronic inflammation in high fat diet challenged C57BL/6J mice is associated with acceleration of age-dependent renal amyloidosisScientific Reports51647410.1038/srep1647426563579PMC4643235
  70. WangL WenP van de LeemputJ ZhaoZ HanZ 2021Slit diaphragm maintenance requires dynamic clathrin-mediated endocytosis facilitated by AP-2, Lap, Aux and Hsc70-4 in nephrocytesCell & Bioscience118310.1186/s13578-021-00595-433975644PMC8111712
  71. WeaversH Prieto-SánchezS GraweF Garcia-LópezA ArteroR Wilsch-BräuningerM Ruiz-GómezM SkaerH DenholmB 2009The insect nephrocyte is a podocyte-like cell with a filtration slit diaphragmNature45732232610.1038/nature0752618971929PMC2687078
  72. WenP ZhangF FuY ZhuJY HanZ 2020Exocyst genes are essential for recycling membrane proteins and maintaining slit diaphragm in Drosophila NephrocytesJournal of the American Society of Nephrology311024103410.1681/ASN.201906059132238475PMC7217423
  73. WoodcockKJ KierdorfK PouchelonCA VivancosV DionneMS GeissmannF 2015Macrophage-derived upd3 cytokine causes impaired glucose homeostasis and reduced lifespan in Drosophila fed a lipid-rich dietImmunity4213314410.1016/j.immuni.2014.12.02325601202PMC4304720
  74. YamoutH LazichI BakrisGL 2014Blood pressure, hypertension, RAAS blockade, and drug therapy in diabetic kidney diseaseAdvances in Chronic Kidney Disease2128128610.1053/j.ackd.2014.03.00524780456
  75. ZhangF ZhaoY ChaoY MuirK HanZ 2013aCubilin and amnionless mediate protein reabsorption in Drosophila NephrocytesJournal of the American Society of Nephrology2420921610.1681/ASN.201208079523264686PMC3559489
  76. ZhangF ZhaoY HanZ 2013bAn in vivo functional analysis system for renal gene discovery in Drosophila Pericardial NephrocytesJournal of the American Society of Nephrology2419119710.1681/ASN.201208076923291470PMC3559487
  77. ZhangH NairV SahaJ AtkinsKB HodginJB SaundersTL MyersMGJr WernerT KretzlerM BrosiusFC 2017Podocyte-specific JAK2 overexpression worsens diabetic kidney disease in miceKidney International9290992110.1016/j.kint.2017.03.02728554737PMC5610635
  78. ZhaoY CoccoC DomenichiniS SamsonML RabinowL 2015The IMD innate immunity pathway of Drosophila influences somatic sex determination via regulation of the Doa locusDevelopmental Biology40722423110.1016/j.ydbio.2015.09.01326434917
  79. ZhaoF ZhuJY RichmanA FuY HuangW ChenN PanX YiC DingX WangS WangP NieX HuangJ YangY YuZ HanZ 2019Mutations in NUP160 are implicated in steroid-resistant nephrotic syndromeJournal of the American Society of Nephrology3084085310.1681/ASN.201808078630910934PMC6493979
  80. ZhaoY KhallafMA JohanssonE DzakiN BhatS AlfredssonJ DuanJ HanssonBS KnadenM AleniusM 2022Hedgehog-mediated gut-taste neuron axis controls sweet perception in DrosophilaNature Communications13781010.1038/s41467-022-35527-4PMC976335036535958
  81. ZhaoY DuanJ van de LeemputJ HanZ 2023aCardiac Neurons Expressing a Glucagon-like Receptor Mediate Cardiac Arrhythmia Induced by High-Fat Diet in DrosophilabioRxiv10.1101/2023.12.13.571403PMC1296571741789689
  82. ZhaoY JohanssonE DuanJ HanZ AleniusM 2023bFat- and sugar-induced signals regulate sweet and fat taste perception in DrosophilaCell Reports4211338710.1016/j.celrep.2023.11338737934669PMC11212107
  83. ZhaoY DuanJ HanID van de LeemputJ RayPE HanZ 2025Piezo, nephrocyte function, and slit diaphragm maintenance in DrosophilaJournal of the American Society of Nephrology3639340510.1681/ASN.000000052939431457PMC11888958
  84. ZhuJ FuY RichmanA ZhaoZ RayPE HanZ 2017A personalized model of COQ2 nephropathy rescued by the wild-type COQ2 allele or dietary coenzyme Q10 supplementationJournal of the American Society of Nephrology282607261710.1681/ASN.201606062628428331PMC5576924
  85. ZhuangS ShaoH GuoF TrimbleR PearceE AbmayrSM 2009Sns and Kirre, the Drosophila orthologs of Nephrin and Neph1,direct adhesion, fusion and formation of a slit diaphragm-like structure in insect nephrocytesDevelopment1362335234410.1242/dev.03160919515699PMC2729346

Author Contributions

  • Rosa Bianchi: Funding acquisition, Writing – review & editing, Supervision, Writing – original draft
  • Wei Hayashi: Methodology, Conceptualization, Writing – original draft

Declarations

Funding

Funding was provided by the European Research Council under the European Union’s Horizon Europe programme (grant agreement 101054321).

Competing Interests

The authors declare no competing interests.

Data Availability

Source code and data are available at a public repository; the link is provided in the Methods section.

Demonstration content: the full text of this article is adapted from “JAK-STAT pathway activation compromises nephrocyte function in a Drosophila high-fat diet model of chronic kidney disease” by Zhao and colleagues (eLife, 2025; https://doi.org/10.7554/eLife.96987), which is available under a Creative Commons license (https://creativecommons.org/licenses/by/4.0/).

Download
PDFJATS XML