Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

Extra-cellular matrix induced by steroids through a G-protein coupled receptor in a Drosophila model of renal fibrosis

Wenjing Zheng, Karen Ocorr, View ORCID ProfileMarc Tatar
doi: https://doi.org/10.1101/653329
Wenjing Zheng
1Department of Ecology and Evolutionary Biology, Division of Biology and Medicine, Brown University, Providence RI 02912
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Karen Ocorr
2Development, Aging and Regeneration Program, SBP Medical Discovery Institute, La Jolla, CA 92037
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Marc Tatar
1Department of Ecology and Evolutionary Biology, Division of Biology and Medicine, Brown University, Providence RI 02912
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Marc Tatar
  • For correspondence: Marc_Tatar@Brown.edu
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Supplementary material
  • Preview PDF
Loading

Abstract

Aldosterone is produced by the mammalian adrenal cortex to modulate blood pressure and fluid balance, however excessive, prolonged aldosterone production promotes fibrosis and kidney failure. How aldosterone triggers disease may involve actions that are independent of its canonical mineralocorticoid receptor. Here we present a Drosophila model of renal pathology caused by excess extra-cellular matrix formation, stimulated by exogenous aldosterone and insect ecdysone steroids. Chronic administration of aldosterone or ecdysone induces expression and accumulation of collagen-like pericardin at adult nephrocytes – podocyte-like cells that filter circulating hemolymph. Excess pericardin deposition disrupts nephrocyte (glomerular) filtration and causes proteinuria in Drosophila, hallmarks of mammalian kidney failure. Steroid-induced pericardin arises from cardiomyocytes associated with nephrocytes, reflecting an analogous role of mammalian myofibroblasts in fibrotic disease. Remarkably, the canonical ecdysteroid nuclear hormone receptor, ecdysone receptor EcR, is not required for aldosterone or ecdysone to stimulate pericardin production or associated renal pathology. Instead, these hormones require a cardiomyocyte-associated G-protein coupled receptor, dopamine-EcR (dopEcR), a membrane-associated receptor previously characterized in the fly brain as affecting behavior. This Drosophila renal disease model reveals a novel signaling pathway through which steroids may potentially modulate human fibrosis through proposed orthologs of dopEcR.

Significance Statement Aldosterone regulates salt and fluid homeostasis, yet excess aldosterone contributes to renal fibrosis. Aldosterone acts through a nuclear hormone receptor, but an elusive, G-protein coupled receptor (GPCR) is thought to also mediate the hormone’s pathology. Here we introduce a Drosophila model of renal fibrosis. Flies treated with human aldosterone produce excess extra-cellular matrix and that causes kidney pathology. Flies treated with the insect steroid ecdysone produce similar pathology, and from this analogous response we identify an alternative receptor through which steroids mediate renal fibrosis -- the GPCR dopamine-Ecdysone Receptor (dopEcR). dopEcR functions in heart muscle cells associated with nephrocytes, analogous to the role of myofibroblasts in human fibrosis. This finding opens avenues to identify mammalian GPCR homologs of dopEcR through which aldosterone mediates renal fibrosis.

Introduction

Aldosterone is a primary regulator of sodium and potassium homeostasis in kidney distal tubules. But when chronically elevated as in diabetes and primary aldosteronism (1), aldosterone promotes kidney interstitial fibrosis and glomerulosclerosis (2–4). These events are preceded by elevated inflammation through monocytes and macrophage infiltration followed by proliferation of myofibroblasts that secrete fibrinogen, collagens and elastins. Aldosterone increases reactive oxygen species (ROS) to induce profibrotic factors such as Transforming Growth Factor-β1 (TGF-β1), Plasminogen Activator Inhibitor-1, and Enothelin-1 (4). TGF-β1 contributes to fibrosis by activating myofibroblasts (5) as well as through suppressing matrix metalloproteinases, which can further promote excess extra-cellular matrix (6). Aldosterone affects these processes through its interaction with the mineralocorticoid nuclear hormone receptor (MR), as inferred from studies where blockade of MR activity prevents aldosterone-associated inflammatory and fibrotic outcomes (7–9).

Many data also suggest that aldosterone contributes to fibrosis through rapid signaling independent of MR (4). Aldosterone enhances TGF-β1 expression and fibrosis in part through stimulation of ERK1/2 that cannot be blocked by spironolactone, (10–12), while aldosterone fosters hypertrophy in cardiomyocytes through action on ERK5 and PKC (13). As well, aldosterone effectively induces calcium influx in fibroblasts that are derived from MR-deficient mice (14). Angiotensin receptors crosstalk with MR to modulate NF-κB in vascular smooth muscle cells (VSMC) stimulated with aldosterone (15), suggesting that aldosterone may act through G-protein-coupled receptors (GPCR). With considerable debate, GPER1 has been proposed as an alternative GPCR for aldosterone (16–19). In VSMC, aldosterone was seen to activate PI3 kinase and ERK through both GPER1 and MR (20). Emerging evidence, however, shows that 17β-estradiol is the steroid agonist of GPER1 (21–23), and no pharmacological evidence demonstrates GPER1 to interact with aldosterone. The problem remains: through which receptor aside from MR might aldosterone stimulate G-coupled signaling and how does this modulate fibrosis?

Here we address these issues with a new model of steroid-induced fibrosis based on Drosophila melanogaster. Genetic data reveal the GPRC dopamine-EcR (dopEcR) is necessary and sufficient for aldosterone and insect ecdysone to induce excess extra cellular matrix at nephrocytes and to disrupt fly renal function. Based on our findings we propose that mammalian homologs of dopEcR may offer a novel entrée to moderate fibrotic pathology in humans.

Results

The tubular heart of adult Drosophila is surrounded by a collection of pericardial cells, podocyte-like nephrocytes that conduct size-selective filtration of hemolymph (24, 25). The heart tube and the associated nephrocytes are enmeshed in an extracellular matrix composed of collagen-like proteins including pericardin (collagen IV) (26, 27). In a first step to develop a model of Drosophila renal fibrosis we quantified nephrocyte function my measuring levels of protein in excreta (frass) as an analog to proteinuria seen in humans with glomerular dysfunction (28). Frass arises as a by-product of digestion and from discharge of Malpighian renal tubules. Frass quality is modulated in response to diet, mating and internal metabolic state (29), and in response to activity of nephrocytes (30, 31). We collected frass from adult males in microcentrifuge tubes and assayed for total protein content normalized to uric acid as a measure of excretion volume. Diets of high sugar or salt decreased protein excretion compared to normal diet (Fig 1A). Protein in frass was elevated in adults fed aldosterone for two weeks (Fig 1B) yet not when fed aldosterone for only 24 h (Fig S1). To determine if nephrocytes contribute to frass protein content, we depleted nephrocyte slit diaphragm genes kirre and sticks-n-stones (sns), which encode homologs of mammalian nefrin. Previous reports show that reduced kirre and sns impairs nephrocyte filtration measured by uptake of fluoro-dextran beads (25, 32). We replicate this result (Fig 1D, E) and subsequently observed that reduced kirre and sns also elevates protein excretion (Fig 1C). Thus, defects in nephrocyte function are sufficient to induce proteinuria in Drosophila.

Figure 1.
  • Download figure
  • Open in new tab
Figure 1. Aldosterone and ecdysone induce renal dysfunction in adult Drosophila.

A) Proteinuria in 3 w old males measured as excreted protein/uric acid in adults fed high salt diet and high sugar diet, assessed across four independent wildtype backgrounds, each with 4-5 biological replicates. Values normalized to control treatment within each background. B) Proteinuria in 3 w old males fed for two weeks with 20-hydroxyecdysone (20E), ecdysone (E) or aldosterone, assessed with three wildtype backgrounds, each with 4-5 biological replicates. C) Proteinuria in 3 w old males expressing RNAi in nephrocytes to deplete slit diaphragm encoding kirre or sns. D) Confocal images (representative z-stack) of nephrocytes of 3 w old females in ex vivo dextran-bead filtration assay. Efficient filtration presented by wildtype; impaired filtration occurs with depletion of slit diaphragm (sns-RNAi) and by treatment of wildtype with aldosterone or ecdysone. E, F) Quantification of fluorescence intensity from biological replicates of nephrocytes in ex vivo dextran-bead filtration assay when slit diaphragm is depleted by RNAi, and wildtype adults are treated with 20E, ecdysone or aldosterone. (A-C, E, F: ANOVA with Dunnett’s post hoc comparison to control, * p < 0.05, ** p < 0.001. Plots show means with SD.) G) Survival upon instant diet supplemented with NaCl (1.5%) for cohorts (each N= 230-330) while treated with 20E, ecdysone, or aldosterone relative to control. Survival was reduced in each treatment, pair-wise contrasts to control, log-rank test, p < 0.001. H) Survival upon instant diet for cohorts (each N= 216-280) while treated with 20E, ecdysone, or aldosterone. Relative to control (median life span = 42 d), survival was increased by 20E (median life span = 50 d; log-rank test, p = 0.051), but not significantly affected by aldosterone (median lifespan = 48 d, log-rank test, p = 0.742) or ecdysone (median lifespan = 46 d, log-rank test, p = 0.185).

Drosophila do not synthesize aldosterone, a mammalian steroid hormone produced in the renal cortex. Aldosterone may act in Drosophila either as a mimic of insect steroids or by providing a precursor in the synthesis of insect steroids. Notably, 20-hydroxyecdyone (20E) is the primary active steroid in Drosophila. 20E is oxidized from prohormone ecdysone by 20-hydroxylase (encoded by shade) at target cells. 20E activates the nuclear hormone ecdysone receptor (EcR) to modulate transcription. Feeding adults 20E for two weeks did not stimulate proteinuria, but proteinuria was elevated in adults chronically fed ecdysone (Fig 1B). Likewise, chronic aldosterone and ecdysone, but not 20E, suppressed dextran filtration by nephrocytes (Fig 1F). While aldosterone and ecdysone affect nephrocyte function and associated proteinuria, all tested steroids (aldosterone, ecdysone and 20E) reduced the survival of adults on high salt diet (Fig 1G). We found no consistent association between exogenous steroids, renal function and survival of adults on normal diet (Fig 1H).

The extracellular matrix surrounding pericardial nephrocytes consists of collagen-like proteins including pericardin, col4a1 and Viking (26, 27, 33). Pericardin (prc) mRNA was induced by overnight feeding with aldosterone and ecdysone, but not by 20E (Fig 2A). Neither col4a1 nor Viking mRNA were induced by any of the tested steroids (Fig 2B, C). Despite induction of prc mRNA, overnight steroid feeding itself did not elevate proteinuria (Fig S1). In contrast, aldosterone and ecdysone fed to wildtype adults for two weeks stimulated elevated pericardin protein deposition in the nephrocyte extracellular matrix, while no effect was seen with 20E (Fig 2D, E). Depletion of pericardin mRNA from cardiomyocytes (tinΔ4- gal4>prc(RNAi)) but not nephrocytes (sns-gal4>prc(RNAi)) blocked the ability of aldosterone and ecdysone to induce excess pericardin deposition (Fig 2D, E). Furthermore, pericardin stimulated by aldosterone and ecdysone is sufficient to produce proteinuria and impair nephrocyte filtration. Depletion of prc mRNA from cardiomyocytes blocks the ability of aldosterone and ecdysone to induce pathology, while driving prc(RNAi) in nephrocytes does not (Fig 2F-K). Thus, cardiomyocytes are the source of pericardin protein that accumulates in response to chronic exposure to aldosterone and ecdysone, and impairs nephrocyte function.

Figure 2.
  • Download figure
  • Open in new tab
Figure 2. Pericardin from cardiomyocytes induced by steroids produces renal dysfunction.

A) Pericardin (prc) mRNA in nephrocyte-cardiac tissue induced by ecdysone (E) and aldosterone, but not by 20-hydroxyecdysone (20E). B, C) Collagen-4a1 (col4a1) and Viking mRNA in nephrocyte-cardiac tissue are not induced by steroid hormones. D) Confocal images (representative z-stacks) of nephrocyte-cardiac tissue of 3 w old females after two-week treatment with 20-hydroxyecdysone, ecdysone or aldosterone; wildtype and knock-down genotypes to deplete prc mRNA in nephrocytes (sns-gal4>UAS-prc(RNAi)) and cardiomyocyte (tinΔ4-gal4>UAS-prc(RNAi)). Phalloiden (red) stains cardiomyocyte actin; secondary antibody labels (green) pericardin protein in extra-cellular matrix about nephrocytes and heart. E) Quantification of straining intensity for pericardin ECM protein (each with six independent nephrocyte-heart preparations). F-H) Proteinuria in 3 w old males fed for two weeks with 20-hydroxyecdysone, ecdysone or aldosterone, assessed in wildtype background (yw/UAS-prc(RNAi)), and in genotypes that reduce pericardin (UAS-prc(RNAi)) in nephrocytes (sns-gal4) or cardiomyocytes (tinΔ4-gal4). I-K) Quantification of fluorescence intensity from biological replicates of nephrocytes in ex vivo dextran-bead filtration assay in 3 w old males fed for two weeks with 20-hydroxyecdysone, ecdysone or aldosterone, assessed in wildtype (yw/UAS-prc(RNAi), and in genotypes that reduce pericardin (UAS-prc(RNAi)) in nephrocytes (sns-gal4) or cardiomyocytes (tinΔ4-gal4). (A-C, E-K: One-way ANOVA with Dunnett’s comparison relative to control, * p < 0.05, p < 0.01. Plots show mean with SD)

It is striking that ecdysone but not 20E induces pericardin and associated renal pathology in adult Drosophila. This suggests that prc expression and its accumulation in the ECM can be regulated independent of EcR, the canonical nuclear hormone ecdysone receptor of 20E. Indeed, depletion of EcR by RNAi in cardiomyocytes did not prevent the steroid-dependent induction of prc mRNA, or associated ECM accumulation and renal pathology (Fig 3A, C, E). As an alternative, dopamine-EcR (dopEcR) is a membrane GPCR receptor of ecdysone described to function in the fly brain (34, 35). We found that dopEcR mRNA could be detected in adult pericardial tissue (heart-nephrocytes), and these levels increased after feeding with aldosterone and ecdysone (Fig 3G). Consistent with the notion that dopEcR is required for aldosterone and ecdysone to stimulate renal pathology, depleting dopEcR from cardiomyocytes blocks the ability of aldosterone and ecdysone to induce prc mRNA expression, elevate proteinuria and inhibit nephrocyte filtration (Fig 3B, D, F). Likewise, dopEcR in cardiomyocytes is required for aldosterone and ecdysone to induce excess pericardin protein production. Whereas, both hormones were able to stimulate pericardin production in flies with nephrocyte-specific dopEcR knockdown (sns-Gal4>dopEcR(RNAi)), or in flies with nephrocyte-specific and cardiac-specific EcR knockdown (sns-Gal4>EcR(RNAi) and tinΔ4-Gal4>EcR(RNAi)) (Fig 3I, J, K).

Figure 3.
  • Download figure
  • Open in new tab
Figure 3. Cardiomyocyte dopEcR required for steroid induction of fibrosis and renal pathology.

A, C, E) Depletion of nuclear hormone receptor EcR by RNAi does not block ecdysone and aldosterone induction of nephrocyte-cardiac tissue prc mRNA (A), proteinuria (C) or loss of nephrocyte dextran filtration (E). B, D, F) Depletion of GPCR dopEcR by RNAi blocks ecdysone and aldosterone induction of nephrocyte-cardiac tissue prc mRNA (B), proteinuria (D) and loss of nephrocyte dextran filtration (F). G) dopEcR mRNA occurs and is elevated in nephrocyte-heart pericardial tissue of 3 w old adults treated overnight with ecdysone or aldosterone. H) Confocal images (representative z-stacks) of nephrocyte-cardiac tissue of 3 w old females after two-week treatment as control, or with ecdysone or aldosterone applied to genotypes that deplete EcR or dopEcR mRNA in nephrocytes (sns-gal4) or cardiomyocytes (tinΔ4-gal4). Cardiomyocyte actin stained by phalloiden, red. Pericardin protein of extra-cellular matrix about nephrocytes and heart, green. I, J) Quantification of straining intensity for pericardin ECM protein (each group, six independent nephrocyte-heart preparations) for genotypes to deplete EcR mRNA (I) or dopEcR mRNA (J) in nephrocytes (sns-gal4) or cardiomyocytes (tinΔ4-gal4). (A-G, I, J: One-way ANOVA with Dunnett’s comparison relative to control, * p < 0.05, p < 0.01. Plots show mean with SD.)

Discussion

Derived from cholesterol, mammalian aldosterone is synthesized in the adrenal cortex into a 21-carbon, C21-hydroxyl steroid and controls plasma Na+ and K+, water balance and blood pressure. In insects, ecdysone has a similar structure - a 27-carbon steroid with hydroxyl groups at C21 and C27. In larvae ecdysone is secreted from the prothoracic gland and controls insect development through its action as 20E via the nuclear hormone receptor EcR. Our data show that both aldosterone and ecdysone fed to adult Drosophila stimulate the expression and accumulation of pericardin in cardiac associated extracellular matrix, and to produce renal dysfunction akin to human fibrosis. In these adults, aldosterone and ecdysone appear to function through the G-protein coupled receptor dopEcR, and not through the canonical nuclear hormone receptor EcR, the otherwise typical pathway by which ecdysteroids regulate insect development and physiology. How aldosterone mimics ecdysone in this context remains unknown. Future work will be needed to determine if aldosterone itself binds to dopEcR, as was shown for Ponasterone A and ecdysone (36), or whether it simply acts as a precursor molecule that can be converted to ecdysone within Drosophila. It also remains an open question to determine what roles ecdysone normally plays in development through its control of pericardin, for instance as it might affect heart remodeling during molts and pupation (37).

Although ecdysone is synthesized in the prothoracic gland during development and in adult female follicles, adult somatic tissue including Malpighian tubules also produce ecdysone in response to desiccation stress (38). Ecdysone circulating in adult hemolymph may act at many sites aside from its classic targets of fat body and ovary (39). Notably, dopEcR functions in the fly brain as an alternative ecdysone receptor (34, 35). Here we document dopEcR is required in cardiomyocytes to modulate steroid-induced fibrosis. Fibrosis in humans arises from myofibroblasts that secrete extracellular matrix proteins including fibronectins, elastins and collagens (40–42). In Drosophila, we show that pericardin appears to be expressed by cardiomyocytes, suggesting these cells have an analogous function to mammalian myofibroblasts. We also show that chronic induction of pericardin by steroid hormones acting at cardiomyocytes stimulates excess ECM accumulation, proteinuria and nephrocyte filtration defects.

DopEcR is a dual agonist receptor (43). In neurons, DopEcR transduces signals from both dopamine and ecdysone to regulate mating behavior and ethanol sensitivity (34, 35). Activation by dopamine induces cAMP-mediated signal transduction. Ecdysone has greater affinity to dopEcR and through unknown mechanisms will displace dopamine and induce alternative signal transduction mediated by MAP kinases (36). Reports are mixed on whether ecdysone also affects cAMP via DopEcR because dopamine alone increased cAMP in Sf9 cells expressing dopEcR (35, 36). In mammalian cells, cAMP can induce PKA to phosphorylate CREB, which then localizes to promoters. Human CREB targets include several collagen genes associated with extracellular matrix (44, 45), and cAMP stimulation suppresses collagen-I expression in a CREB dependent manner (46). Accordingly, we hypothesize that dopamine-cAMP-associated transduction in response to dopEcR may negatively regulate pericardin. If so, dopamine in the absence of steroid hormones may suppress ECM accumulation.

In contrast, ecdysone stimulated DopEcR can signal through dEGFR to ERK1/2 as seen in transfected Sf9 cells and in a neuronal analysis of ethanol induced sedation (34, 36). In mammals, EGFR signaling is broadly implicated in renal fibrosis (47), and these effects may be modulated in part by GPCR crosstalk (48). MAPK/ERK modulates TGF-β1 and its transcription factor Smad2/3, which potently induces collagen transcription in fibrosis (49). Future work can resolve whether aldosterone and ecdysone in Drosophila uses dopEcR to license the ability of dEGFR to stimulate pericardin.

Studies in mammals suggest aldosterone may also signal via a membrane associated GPCR. GPER1 has been proposed to function as a non-genomic aldosterone receptor (21, 22, 50). GPER1-dependent effects induced by aldosterone are reported in various models including renal cortical adenocarcinoma cells (17), and are inferred from mouse models with tissue specific mineralocorticoid receptor gene deletion (51). However, no data establish the mechanism of non-genomic action for aldosterone through GPER1 (23, 52). Furthermore, the leading steroid candidate for GPER1 is 17β-estradiol (53), and the GPER-dependent impact of aldosterone in cells could reflect heterologous desensitization. Alternative dopEcR homologs can be identified in the human genome using the DIOPT Ortholog Prediction Tool, including GRP52 (sequence similarity 46%) and UTS2R (sequence similarity 44%). GPR52 is an orphan G-protein coupled receptor described to modulate Huntingtin protein (HTT) through cAMP-dependent mechanisms (54). Knockdown of Gpr52 reduces HTT levels in a human tissue model, whereas neurodegeneration is suppressed by knockdown of dopEcR in transgenic Drosophila that express human Htt. The Urotensin II receptor (UTS2R) is a conserved GPCR implicated to function in renal fibrosis by trans-modulating EGFR and activating MAPK (55, 56). In an induced model of rat diabetes, kidneys expressed elevated Urotensin II, and UTS2R was required for exogenous Urotensin to induce TGF-β1 and ECM collagen. These candidates illustrate the translational potential of the Drosophila steroid-induced model of fibrosis. Understanding how dopEcR modulates fibrosis in Drosophila will uncover how related signaling elements affect fibrosis in humans.

Material and methods

Fly stocks

Unless noted, wildtype flies were yw (ywR). TinΔ4-Gal4 was a gift from the Manfred Frasch laboratory (57). sns-Gal4 was obtained from the Bloomington Stock Center (Stock #76160) and UAS-pericardin(RNAi) was obtained from the Vienna Drosophila Research Center (Stock #GD 41321). UAS-EcR(RNAi) was from the laboratory of Neal Silverman (UMass Medical). dopEcR(RNAi) is kk103494 of VDRC.

Steroid and diet treatment

Ecdysone (Sigma-Aldrich #E9004), 20-Hydroxyecdysone (Sigma-Aldrich #H5142) and Aldosterone (Sigma-Aldrich #A9477) were dissolved in ethanol at 5mg/ml. Flies were reared in bottles with emerging adults permitted to mate for 2-3. Adult were then separated by sex into 1L demography cages at ~ 120 adults per cage. Adults were fed standard laboratory cornmeal-yeast-sugar diet until age 7-10 days, at which time food media was switched to 0.5g Genesee Scientific instant fly media (Genesee Scientific #66-117) hydrated with 2ml of water containing vehicle control (150 ul ethanol) or vehicle with 150 ul of hormone solution. For chronic exposure to steroids, flies were treated for the next 14 days at 25 °C fly with media vials changed every 3 days. For overnight exposure to steroids, flies were maintained in demography cages with untreated instant fly media until age 20 days old, then exposed to diets with appropriate hormone conditions for 24 hours. In all trails, renal traits and prc mRNA were assessed in adults at 3 weeks old. The same protocols were used to expose adults to high salt or high sugar, where instant media was moistened with water containing 1.5% NaCl. To vary dietary glucose, adults were aged to 3 weeks on otherwise standard lab diet where glucose was set at 5% (control, normal) or at 34% (high sugar diet).

Proteinuria

For each biological replicate, frass of 15-20 males was collected for 2.5 hours in a 1.5ml centrifuge tube covered with a breathable foam plug, at 25 °C. Deposited frass was fully dissolved with 20ul 1xPBS, providing 10ul to assess total protein and 10ul to measure uric acid, which serves as a proxy for the quantity of deposited frass. Total urine protein was determined by Pierce BCA Protein assay (Thermo Scientific #23227). Uric acid was measured by QuantiChrom Uric Acid Assay (Bioassay systems, DIUA-250).

Immunohistology

Nephrocyte-heart tissue from 3 w old adults were dissected in PBS, fixed with 4% formaldehyde in PBS for 30 minutes and washed three times for 10 minutes with PBTA (1xPBS,1.5% BSA, 0.3% Tween20) at room temperature. The washed tissue was incubated with 100 ul primary antibody (mouse anti-Pericardin 1:100, Developmental Studies Hybridoma Bank) diluted in PBTA overnight at 4C, washed 3×10 minutes with 1ml PBTA at room temperature, then incubated in secondary antibody (goat anti-mouse Alexa488 1:200, Alexa555-phalloidin 1:100, ThermoFisher Scientific) diluted in PBTA overnight, washed 3×10 minutes with 1ml PBTA at room temperature, and mounted. Confocal images were obtained with a Zeiss 800 and quantified by imageJ software. The full length of pericardial tissue was imaged from all samples at 488 nm with the same laser intensity setting to produce a Z-stack comprised of 46 optical slices.

Nephrocyte filtration

Adult nephrocyte-heart tissue was dissected in ADH (Artificial Drosophila Hemolymph, 108 mM Na+, 5 mM K+, 2 mM Ca2+, 8 mM MgCl2, 1 mM NaH2PO4, 4 mM NaHCO3, 10 mM sucrose, 5 mM trehalose, 5 mM Hepes, pH 7.1), incubated at 25°C for 15 minutes with AlexaFluor568-Dextran (10,000 MW, Life Technology) diluted in ADH at a concentration of 33mg/ml, washed 3×10 minutes with cold PBS at 4C, then fixed in 4% formaldehyde for 10 minutes at room temperature, washed 3×10 minutes with PBS at room temperature, and mounted in PBS. Confocal images were obtained with a Zeiss 800 and quantified by imageJ software.

Acknowledgements

WZ and MT received support from NIH grant PO1 AG033561, NIDDK Diabetic Complications Consortium grant DK076169, and the office of the Dean of Biology and Medicine, Warren Alpert School of Medicine, Brown University. KO received support from NIH grant R01 HL132241. Erika Taylor assisted KO at the SBP Medical Discovery Institute.

References

  1. 1.↵
    Nagase M & Fujita T (2011) Endocrinological Aspects of Proteinuria and Podocytopathy in Diabetes: Role of the Aldosterone/Mineralocorticoid Receptor System. Current diabetes reviews 7(1):8–16.
    OpenUrl
  2. 2.↵
    Azibani F, Fazal L, Chatziantoniou C, Samuel JL, & Delcayre C (2013) Aldosterone mediates cardiac fibrosis in the setting of hypertension. Curr Hypertens Rep 15(4):395–400.
    OpenUrlCrossRefPubMed
  3. 3.
    Ibrahim HN & Hostetter TH (2003) Aldosterone in renal disease. Current opinion in nephrology and hypertension 12(2):159–164.
    OpenUrlCrossRefPubMedWeb of Science
  4. 4.↵
    Brown NJ (2013) Contribution of aldosterone to cardiovascular and renal inflammation and fibrosis. Nat Rev Nephrol 9(8):459–469.
    OpenUrlCrossRefPubMed
  5. 5.↵
    Barnes JL & Gorin Y (2011) Myofibroblast differentiation during fibrosis: role of NAD(P)H oxidases. Kidney international 79(9):944–956.
    OpenUrlCrossRefPubMedWeb of Science
  6. 6.↵
    Zhao H, et al. (2013) Matrix metalloproteinases contribute to kidney fibrosis in chronic kidney diseases. World J Nephrol 2(3):84–89.
    OpenUrlCrossRefPubMed
  7. 7.↵
    Tesch GH & Young MJ (2017) Mineralocorticoid Receptor Signaling as a Therapeutic Target for Renal and Cardiac Fibrosis. Front Pharmacol 8:313.
    OpenUrl
  8. 8.
    Ibarrola J, et al. (2018) Aldosterone Impairs Mitochondrial Function in Human Cardiac Fibroblasts via A-Kinase Anchor Protein 12. Scientific reports 8(1):6801.
    OpenUrl
  9. 9.↵
    Montes-Cobos E, et al. (2015) Inducible Knock-Down of the Mineralocorticoid Receptor in Mice Disturbs Regulation of the Renin-Angiotensin-Aldosterone System and Attenuates Heart Failure Induced by Pressure Overload. PLoS One 10(11):e0143954.
    OpenUrl
  10. 10.↵
    Han JS, Choi BS, Yang CW, & Kim YS (2009) Aldosterone-induced TGF-beta1 expression is regulated by mitogen-activated protein kinases and activator protein-1 in mesangial cells. J Korean Med Sci 24 Suppl:S195–203.
    OpenUrlCrossRefPubMed
  11. 11.
    Fu GX, Xu CC, Zhong Y, Zhu DL, & Gao PJ (2012) Aldosterone-induced osteopontin expression in vascular smooth muscle cells involves MR, ERK, and p38 MAPK. Endocrine 42(3):676–683.
    OpenUrlCrossRefPubMed
  12. 12.↵
    Min LJ, et al. (2005) Aldosterone and angiotensin II synergistically induce mitogenic response in vascular smooth muscle cells. Circ Res 97(5):434–442.
    OpenUrlAbstract/FREE Full Text
  13. 13.↵
    Araujo CM, et al. (2016) Rapid effects of aldosterone in primary cultures of cardiomyocytes - do they suggest the existence of a membrane-bound receptor? J Recept Signal Transduct Res 36(5):435–444.
    OpenUrlCrossRefPubMed
  14. 14.↵
    Haseroth K, et al. (1999) Rapid nongenomic effects of aldosterone in mineralocorticoid-receptor-knockout mice. Biochem Biophys Res Commun 266(1):257–261.
    OpenUrlCrossRefPubMedWeb of Science
  15. 15.↵
    Lemarie CA, et al. (2009) Aldosterone-induced activation of signaling pathways requires activity of angiotensin type 1a receptors. Circ Res 105(9):852–859.
    OpenUrlAbstract/FREE Full Text
  16. 16.↵
    Wendler A, Albrecht C, & Wehling M (2012) Nongenomic actions of aldosterone and progesterone revisited. Steroids 77(10):1002–1006.
    OpenUrlCrossRefPubMed
  17. 17.↵
    Feldman RD, et al. (2016) Aldosterone mediates metastatic spread of renal cancer via the G protein-coupled estrogen receptor (GPER). FASEB journal: official publication of the Federation of American Societies for Experimental Biology 30(6):2086–2096.
    OpenUrl
  18. 18.
    Ren Y, et al. (2014) Aldosterone sensitizes connecting tubule glomerular feedback via the aldosterone receptor GPR30. American journal of physiology. Renal physiology 307(4):F427–434.
    OpenUrlCrossRefPubMed
  19. 19.↵
    Brem AS, Morris DJ, & Gong R (2011) Aldosterone-induced fibrosis in the kidney: questions and controversies. American journal of kidney diseases : the official journal of the National Kidney Foundation 58(3):471–479.
    OpenUrl
  20. 20.↵
    Gros R, et al. (2011) GPR30 expression is required for the mineralocorticoid receptor-independent rapid vascular effects of aldosterone. Hypertension 57(3):442–451.
    OpenUrlCrossRef
  21. 21.↵
    Evans NJ, Bayliss AL, Reale V, & Evans PD (2016) Characterisation of Signalling by the Endogenous GPER1 (GPR30) Receptor in an Embryonic Mouse Hippocampal Cell Line (mHippoE-18). PLoS One 11(3):e0152138.
    OpenUrl
  22. 22.
    Barton M & Meyer MR (2015) Nicolaus Copernicus and the rapid vascular responses to aldosterone. Trends Endocrinol Metab 26(8):396–398.
    OpenUrlCrossRefPubMed
  23. 23.↵
    Cheng SB, et al. (2014) Anatomical location and redistribution of G protein-coupled estrogen receptor-1 during the estrus cycle in mouse kidney and specific binding to estrogens but not aldosterone. Molecular and cellular endocrinology 382(2):950–959.
    OpenUrlCrossRefPubMed
  24. 24.↵
    Beyenbach KW, Skaer H, & Dow JA (2010) The developmental, molecular, and transport biology of Malpighian tubules. Annual review of entomology 55:351–374.
    OpenUrlCrossRefPubMedWeb of Science
  25. 25.↵
    Weavers H, et al. (2009) The insect nephrocyte is a podocyte-like cell with a filtration slit diaphragm. Nature 457(7227):322–326.
    OpenUrlCrossRefPubMedWeb of Science
  26. 26.↵
    Chartier A, Zaffran S, Astier M, Semeriva M, & Gratecos D (2002) Pericardin, a Drosophila type IV collagen-like protein is involved in the morphogenesis and maintenance of the heart epithelium during dorsal ectoderm closure. Development 129(13):3241–3253.
    OpenUrlAbstract/FREE Full Text
  27. 27.↵
    Hollfelder D, Frasch M, & Reim I (2014) Distinct functions of the laminin beta LN domain and collagen IV during cardiac extracellular matrix formation and stabilization of alary muscle attachments revealed by EMS mutagenesis in Drosophila. BMC developmental biology 14:26.
    OpenUrl
  28. 28.↵
    Ziyadeh FN & Wolf G (2008) Pathogenesis of the podocytopathy and proteinuria in diabetic glomerulopathy. Current diabetes reviews 4(1):39–45.
    OpenUrl
  29. 29.↵
    Cognigni P, Bailey Ap Fau - Miguel-Aliaga I, & Miguel-Aliaga I (2011) Enteric neurons and systemic signals couple nutritional and reproductive status with intestinal homeostasis. Cell Metabolism 5:92–104.
    OpenUrl
  30. 30.↵
    Zhang F, Zhao Y, Chao Y, Muir K, & Han Z (2013) Cubilin and amnionless mediate protein reabsorption in Drosophila nephrocytes. Journal of the American Society of Nephrology: JASN 24(2):209–216.
    OpenUrl
  31. 31.↵
    Helmstadter M & Simons M (2017) Using Drosophila nephrocytes in genetic kidney disease. Cell and tissue research 369(1):119–126.
    OpenUrl
  32. 32.↵
    Na J, Sweetwyne MT, Park AS, Susztak K, & Cagan RL (2015) Diet-Induced Podocyte Dysfunction in Drosophila and Mammals. Cell reports 12(4):636–647.
    OpenUrl
  33. 33.↵
    Zhang F, Zhao Y, & Han Z (2013) An in vivo functional analysis system for renal gene discovery in Drosophila pericardial nephrocytes. Journal of the American Society of Nephrology: JASN 24(2):191–197.
    OpenUrl
  34. 34.↵
    Petruccelli E, Li Q, Rao Y, & Kitamoto T (2016) The Unique Dopamine/Ecdysteroid Receptor Modulates Ethanol-Induced Sedation in Drosophila. The Journal of neuroscience : the official journal of the Society for Neuroscience 36(16):4647–4657.
    OpenUrlAbstract/FREE Full Text
  35. 35.↵
    Ishimoto H, Wang Z, Rao Y, Wu CF, & Kitamoto T (2013) A novel role for ecdysone in Drosophila conditioned behavior: linking GPCR-mediated non-canonical steroid action to cAMP signaling in the adult brain. PLoS genetics 9(10):e1003843.
    OpenUrl
  36. 36.↵
    Srivastava DP, et al. (2005) Rapid, nongenomic responses to ecdysteroids and catecholamines mediated by a novel Drosophila G-protein-coupled receptor. The Journal of neuroscience : the official journal of the Society for Neuroscience 25(26):6145–6155.
    OpenUrlAbstract/FREE Full Text
  37. 37.↵
    Wilmes AC, Klinke N, Rotstein B, Meyer H, & Paululat A (2018) Biosynthesis and assembly of the Collagen IV-like protein Pericardin in Drosophila melanogaster. Biology open 7(4).
  38. 38.↵
    Zheng W, et al. (2018) Dehydration triggers ecdysone-mediated recognition-protein priming and elevated anti-bacterial immune responses in Drosophila Malpighian tubule renal cells. BMC Biol 16(1):60.
    OpenUrlCrossRef
  39. 39.↵
    Schwedes C, Tulsiani S, & Carney GE (2011) Ecdysone receptor expression and activity in adult Drosophila melanogaster. J Insect Physiol 57(7):899–907.
    OpenUrlCrossRefPubMedWeb of Science
  40. 40.↵
    Tuttle KR, et al. (2014) Diabetic Kidney Disease: A Report From an ADA Consensus Conference. Diabetes Care 37(10):2864.
    OpenUrlAbstract/FREE Full Text
  41. 41.
    Meran S & Steadman R (2011) Fibroblasts and myofibroblasts in renal fibrosis. International journal of experimental pathology 92(3):158–167.
    OpenUrlCrossRefPubMed
  42. 42.↵
    Mack M & Yanagita M (2015) Origin of myofibroblasts and cellular events triggering fibrosis. Kidney international 87(2):297–307.
    OpenUrlCrossRefPubMed
  43. 43.↵
    Evans PD, Bayliss A, & Reale V (2014) GPCR-mediated rapid, non-genomic actions of steroids: comparisons between DmDopEcR and GPER1 (GPR30). Gen Comp Endocrinol 195:157–163.
    OpenUrlCrossRefPubMed
  44. 44.↵
    Zhang Y, et al. (2005) Modification of the Stat1 SH2 domain broadly improves interferon efficacy in proportion to p300/CREB-binding protein coactivator recruitment. J Biol Chem 280(40):34306–34315.
    OpenUrlAbstract/FREE Full Text
  45. 45.↵
    Impey S, et al. (2004) Defining the CREB regulon: a genome-wide analysis of transcription factor regulatory regions. Cell 119(7):1041–1054.
    OpenUrlCrossRefPubMedWeb of Science
  46. 46.↵
    Zhang A (2005) CREB Target Gene Database. (http://natural.salk.edu/CREB/).
  47. 47.↵
    Zhuang S & Liu N (2014) EGFR signaling in renal fibrosis. Kidney international supplements 4(1):70–74.
    OpenUrl
  48. 48.↵
    Yan Y, et al. (2016) Src inhibition blocks renal interstitial fibroblast activation and ameliorates renal fibrosis. Kidney international 89(1):68–81.
    OpenUrlCrossRefPubMed
  49. 49.↵
    Liu F & Zhuang S (2016) Role of Receptor Tyrosine Kinase Signaling in Renal Fibrosis. International journal of molecular sciences 17(5).
  50. 50.
    Feldman RD & Limbird LE (2015) Copernicus Revisited: Overturning Ptolemy’s View of the GPER Universe. Trends Endocrinol Metab 26(11):592–594.
    OpenUrlCrossRefPubMed
  51. 51.↵
    Lother A, Moser M, Bode C, Feldman RD, & Hein L (2015) Mineralocorticoids in the heart and vasculature: new insights for old hormones. Annual review of pharmacology and toxicology 55:289–312.
    OpenUrlCrossRefPubMed
  52. 52.↵
    Rigiracciolo DC, et al. (2016) GPER is involved in the stimulatory effects of aldosterone in breast cancer cells and breast tumor-derived endothelial cells. Oncotarget 7(1):94–111.
    OpenUrlPubMed
  53. 53.↵
    Revankar CM, Cimino DF, Sklar LA, Arterburn JB, & Prossnitz ER (2005) A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 307(5715): 1625–1630.
    OpenUrlAbstract/FREE Full Text
  54. 54.↵
    Yao Y, et al. (2015) A striatal-enriched intronic GPCR modulates huntingtin levels and toxicity. eLife 4.
  55. 55.↵
    Tian L, et al. (2008) Diabetes-induced upregulation of urotensin II and its receptor plays an important role in TGF-beta1-mediated renal fibrosis and dysfunction. American journal of physiology. Endocrinology and metabolism 295(5):E1234–1242.
    OpenUrlCrossRefPubMed
  56. 56.↵
    Vaudry H, et al. (2015) International Union of Basic and Clinical Pharmacology. XCII. Urotensin II, urotensin ll-related peptide, and their receptor: from structure to function. Pharmacological reviews 67(1):214–258.
    OpenUrl
  57. 57.↵
    Lo PC & Frasch M (2001) A role for the COUP-TF-related gene seven-up in the diversification of cardioblast identities in the dorsal vessel of Drosophila. Mech Dev 104(1-2):49–60.
    OpenUrlCrossRefPubMedWeb of Science
Back to top
PreviousNext
Posted May 29, 2019.
Download PDF

Supplementary Material

Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
Extra-cellular matrix induced by steroids through a G-protein coupled receptor in a Drosophila model of renal fibrosis
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Extra-cellular matrix induced by steroids through a G-protein coupled receptor in a Drosophila model of renal fibrosis
Wenjing Zheng, Karen Ocorr, Marc Tatar
bioRxiv 653329; doi: https://doi.org/10.1101/653329
Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Citation Tools
Extra-cellular matrix induced by steroids through a G-protein coupled receptor in a Drosophila model of renal fibrosis
Wenjing Zheng, Karen Ocorr, Marc Tatar
bioRxiv 653329; doi: https://doi.org/10.1101/653329

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Pathology
Subject Areas
All Articles
  • Animal Behavior and Cognition (2647)
  • Biochemistry (5271)
  • Bioengineering (3682)
  • Bioinformatics (15799)
  • Biophysics (7260)
  • Cancer Biology (5629)
  • Cell Biology (8102)
  • Clinical Trials (138)
  • Developmental Biology (4769)
  • Ecology (7524)
  • Epidemiology (2059)
  • Evolutionary Biology (10588)
  • Genetics (7733)
  • Genomics (10138)
  • Immunology (5199)
  • Microbiology (13919)
  • Molecular Biology (5392)
  • Neuroscience (30805)
  • Paleontology (215)
  • Pathology (879)
  • Pharmacology and Toxicology (1525)
  • Physiology (2256)
  • Plant Biology (5026)
  • Scientific Communication and Education (1042)
  • Synthetic Biology (1389)
  • Systems Biology (4150)
  • Zoology (812)