Cyst Reduction by Melatonin in a Novel Drosophila Model of Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) causes progressive cystic degeneration of the renal tubules, the nephrons, eventually severely compromising kidney function. ADPKD is incurable, with half of the patients eventually needing renal replacement. Treatments for ADPKD patients are limited and new effective therapeutics are needed. Melatonin, a central metabolic regulator conserved across all life kingdoms, exhibits oncostatic and oncoprotective activity and no detected toxicity. Here, we used the Bicaudal C (BicC) Drosophila model of polycystic kidney disease to test the cyst-reducing potential of melatonin. Significant cyst reduction was found in the renal (Malpighian) tubules upon melatonin administration and suggest mechanistic sophistication. Similar to vertebrate PKD, the BicC fly PKD model responds to the antiproliferative drugs rapamycin and mimics of the second mitochondria-derived activator of caspases (Smac). Melatonin appears to be a new cyst-reducing molecule with attractive properties as a potential candidate for PKD treatment.


Polycystic Kidney Disease
Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disease affecting 12.5 million people globally. ADPKD typically causes the progressive formation of cysts all along the renal tubules called nephrons [1]. ADPKD has been linked to mutations in the PKD1 gene in about 80% of the cases and in PKD2 in about 15% of patients, with the residual~5% of cases remaining genetically unknown or linked to rare mutations in other loci [1]. Renal cystic disease in general, and ADPKD particularly, display complex metabolic alterations in the tissue of the renal tubule [2] as well as impaired fluid transport [1]. As cysts progressively grow, they compress and damage the surrounding renal parenchyma, impairing the neighboring nephrons, both cystic and non-cystic. The ensuing reduced renal function eventually leads to renal failure in one patient out of two [1].
Considering the wide and potentially beneficial effects of melatonin, as well as its low toxicity profile [107][108][109], we tested the cyst-reducing potential of melatonin utilizing the Drosophila PKD model. The BicC fly model of renal cystogenesis was previously used successfully to test the anti-cystic activity of rapamycin [21] and Smac mimetics [30]. Here, we report that melatonin was found to substantially reduce cysts in the Drosophila PKD model.
Considering the wide and potentially beneficial effects of melatonin, as well as its low toxicity profile [107][108][109], we tested the cyst-reducing potential of melatonin utilizing the Drosophila PKD model. The BicC fly model of renal cystogenesis was previously used successfully to test the anti-cystic activity of rapamycin [21] and Smac mimetics [30]. Here, we report that melatonin was found to substantially reduce cysts in the Drosophila PKD model.

Melatonin Treatment Displayed Regional Specificity in BicC ∆/YC33 Mutants
Similar to human nephrons, the Drosophila renal tubules display regional specialization and differential response to Smac mimics [30]. Thus, we examined the regional physiological response to melatonin treatment. In the BicC ∆/YC33 flies, melatonin appeared to reduce cysts in the proximal, intermediate, and terminal regions of the anterior tubules, respectively, by 59, 37 and 31% (total 27 vs.     Therefore, melatonin administration significantly reduced cysts in BicC ∆/YC33 flies overall, with apparent slightly higher efficacy in the anterior tubule. Moreover, melatonin reduced cysts in the proximal, intermediate and terminal regions of the anterior tubules and in the intermediate and terminal regions of the posterior tubules, with the proximal region displaying a trend toward cyst reduction.

Melatonin Treatment Was Less Efficient in BicC ∆/IIF34 Mutants
The BicC allelic combination, BicC ∆/IIF34 yields more severe defects than BicC ∆/YC33 despite expressing higher levels of a truncated BicC protein [110] and may be a dominant negative [21]. Melatonin was administered to the BicC ∆/IIF34 flies (n = 50) with identical procedure. Melatonin induced an overall trend towards reduced cysts in both anterior and posterior tubules. However, phenotypic variability was such that p-values resulted above significance threshold. Specifically, compared to vehicle-treatment, the anterior tubule displayed 18% less cysts (total 432 vs. 354 cysts, p = 0.1204) and the posterior tubule 7% less (total 503 vs. 469 cysts, p = 0.5329) (Table 1, Figure 3 Figure 3). Therefore, melatonin administration yielded a trend in cyst reduction in the more severely cystic BicC ∆/IIF34 flies; however, p-values remained above significance threshold. Therefore, melatonin administration significantly reduced cysts in BicC Δ/YC33 flies overall, with apparent slightly higher efficacy in the anterior tubule. Moreover, melatonin reduced cysts in the proximal, intermediate and terminal regions of the anterior tubules and in the intermediate and terminal regions of the posterior tubules, with the proximal region displaying a trend toward cyst reduction.

Melatonin Treatment Was Less Efficient in BicC Δ/IIF34 Mutants
The BicC allelic combination, BicC Δ/IIF34 yields more severe defects than BicC Δ/YC33 despite expressing higher levels of a truncated BicC protein [110] and may be a dominant negative [21]. Melatonin was administered to the BicC Δ/IIF34 flies (n = 50) with identical procedure. Melatonin induced an overall trend towards reduced cysts in both anterior and posterior tubules. However, phenotypic variability was such that p-values resulted above significance threshold. Specifically, compared to vehicle-treatment, the anterior tubule displayed 18% less cysts (total 432 vs. 354 cysts, p = 0.1204) and the posterior tubule 7% less (total 503 vs. 469 cysts, p = 0.5329) (Table 1, Figure 3 Figure 3). Therefore, melatonin administration yielded a trend in cyst reduction in the more severely cystic BicC Δ/IIF34 flies; however, p-values remained above significance threshold. Treatments are color-coded: vehicle, left, grey; melatonin, right, green. Melatonin treatment of BicC ∆/IIF34 flies produced a trend in cyst reduction, however, did not significantly differentiate the treated vs. untreated populations.
The BicC PKD fly model recapitulates phenotypic and molecular hallmarks of PKD1-induced PKD [21] and conserved pharmacological response to Smac mimetics [30]. In the mammalian response to pro-apoptotic signals and TNF-α/TNF receptor (TNFR) activation, the Smac protein is released from the mitochondria, which activates the caspase cascade [124]. Smac mimicry has been exploited in oncology to induce apoptosis in TNF-α-dependent cancers [125,126]. Administration of small peptide mimetics of the Smac has been shown to be sufficient to activate the caspase cascade and mitigate cancer [125,126]. Both ADPKD patients and the Pkd1 −/− mouse display high TNF-α amounts in the cystic fluid; moreover, the cystic cells feature higher-than-normal expression of the TNFR1 receptor. Unlike other tubular cells, the cyst-lining cells have abundant TNFR and are bathed in TNF-α-rich fluid, which is thought to fuel an autoactivating loop promoting cyst growth [32,127]. Because of such specific and constitutive TNF-α activation in the cells of ADPKD cysts, one Smac mimetic has been tested in a murine model to preferentially eliminate cystic cells while sparing the non-cystic tubular portions [32]. TNF signaling is highly conserved in Drosophila [128][129][130][131]. We showed that, similar to the ADPKD mouse, administration of four novel Smac mimetics to the BicC fly PKD model significantly reduced cysts in the renal tubules [30]. This underscores the conservation of cystic pathways between human and Drosophila. Interestingly, melatonin has been shown to decrease TNF-α expression [132][133][134][135], which raises the possibility that melatonin pleiotropic functions may contribute to renal tubule normalization. In further support of this possibility, melatonin is a known antiproliferative that normalizes several overactive pathways in both cancer and ADPKD, e.g., ERK, mTOR, PI3K/Akt, PKC [1,16,53,58,71,[136][137][138][139][140][141][142][143]. Note, the BicC fly model of PKD also exhibits hyperactive mTOR [21].
In 1861, it was reported that renal physiology has circadian rhythmicity [144]. Excretion of water, urea and electrolytes all follow 24-h cyclicity [145,146]. Defective glomerular filtration in patients with CKD may disturb sleep (reviewed in [113]). In end-stage renal disease (ESRD), the severity of insomnia appeared to correlate directly with disease progression [112], and inversely with melatonin levels [147][148][149][150][151][152][153]. While specific knowledge of the ADPKD situation is limited, deteriorating kidney function reduces circadian rhythm amplitude [112], suggesting that patients in late stage ADPKD may also have reduced melatonin level and/or function. Melatonin has only been tested in two small clinical trials to treat CKD-related sleep disturbances and the results of one such trials have been published [154]. Small trial NCT04336566 was designed to test melatonin effects on renal function in CKD, however, its results have not been made public. Melatonin pre-treatment has also been found to potentiate the Molecules 2020, 25, 5477 7 of 16 beneficial anti-apoptosis, anti-oxidation, anti-inflammation effects of mesenchymal stem cell (MSC) therapy to treat acute kidney injury (AKI) and CKD ( [155], reviewed in [156]). In a rat CKD model, melatonin pre-treated MSCs also reduced fibrosis in the kidney [135,156]. Together, this evidence strongly suggests that melatonin is crucial for maintaining proper kidney function and that nephrological diseases compromising renal capacity appear to upset the melatonin-dependent pathways.
In the Drosophila PKD model, nightly administration of 150 µM melatonin efficiently reduced cysts in the BicC ∆/YC33 mutants, compared to vehicle-treated flies. These effects were observed both along the entire Malpighian tubules, and regionally. Melatonin reduced cysts in the anterior and posterior tubules by over 30% (p < 0.012). In the terminal, intermediate and proximal regions of the anterior tubules of BicC ∆/YC33 flies, melatonin reduced cysts by 31-59% (p < 0.039). In the posterior tubules, melatonin treatment reduced cysts in the terminal and intermediate regions by 25 and 30% respectively (p < 0.045), while showing a trend towards reduction in the proximal region. Such differential response to melatonin is expected to be rooted onto the functional and physiological differences documented for the anterior and posterior tubules and the tubule regional specializations ( [157]; reviewed in [27]). This property is also consistent with our previous observations of regionally distinct effects of Smac mimetics in the same PKD fly model [30]. In contrast to the BicC ∆/YC33 mutants in which melatonin significantly reduced renal cysts, BicC ∆/IIF34 flies only showed a trend of regional cyst reduction. Notably, the BicC ∆/IIF34 genotype may be dominant negative [21]. This suggests that the genotype may influence the extent of melatonin response of the cystic renal tubule. Future investigations will decipher how melatonin may reduce renal cysts and how specific BicC mutations may affect the melatonin cyst-reducing activity in the renal tubule.
Several core cellular pathways are disrupted in ADPKD. As disease progresses, physiological compensation (e.g., through vasopressin signaling) compounds cellular changes [1]. The molecular detail of PKD pathology is largely unknown [24]. Melatonin biological activity as a potential ameliorator of PKD cystic pathology is intriguing. Firstly, its low toxicity is especially attractive for the long-term management of chronic PKD. Second, melatonin is a master cellular regulator conserved throughout evolution with pleiotropic functions that may help normalize several dysregulated pathways in PKD, e.g., oxidative stress, cell proliferation, fibrosis, renal circadian functions. Tolvaptan, a vasopressin V2 receptor antagonist, appears to primarily target vasopressin-dependent compensation in PKD through mechanisms conserved among mammals. Melatonin may potentially be combined with tolvaptan to reduce toxicity and treat PKD. In oncology, melatonin has been found to potentiate several chemotherapeutics, while simultaneously protecting the patient from their ill effects (e.g., [158][159][160][161][162][163][164][165][166][167][168]). If such property is conserved to its cyst-reducing activity, melatonin may become a prospective candidate for utilization as a single or combination drug in PKD therapy.

Fly Lines and Husbandry
BicC mutants were generated as in [21,30]. Briefly, virgin female flies harboring a BicC deletion in trans to the CyO balancer chromosome, Df(2L)RA5/CyO (obtained from the Bloomington Drosophila Stock Center) were crossed with males carrying one of two hypomorphic BicC mutations in trans to CyO, namely BicC YC33 /CyO and BicC IIF34 /CyO. BicC mutants were selected as the straight-winged progeny with genotypes Df(2L)RA5/BicC YC33 (hereby BicC ∆/YC33 ) and Df(2L)RA5/BicC IIF34 , (hereby BicC ∆/IIF34 ). Such BicC allelic combinations produce truncated proteins and sterile BicC flies [21]. The BicC ∆/IIF34 genotype yields a more severe cystic phenotype than the BicC ∆/YC33 combination and may be dominant negative [21]. Eclosed adult flies were collected every two days to generate 0-2-day old populations to be used in the assays.

Cystic Index
The cystic analysis was performed as in [30], with the following modifications. The 0-2-day old BicC mutant females were housed in vials containing 2 mL cornmeal food (Jazzmix, Fisher Scientific) that were replaced every three days to ensure freshness. During daytime, flies were fed plain cornmeal food. In the evening, flies were transferred into identical vials to which equal volumes (50 µL) of either vehicle (ethanol, control) or 150 µM melatonin (resuspended in ethanol) were added and then incubated overnight. In the morning, flies were transferred to vials with plain cornmeal food. The timing of dissection of the Malpighian tubules for melatonin efficacy was determined empirically with ten female flies micro-dissected at 8, 12, 18, and 25 days after treatment beginning and 18 days post-treatment was chosen for further analyses. This corresponds to fly populations of individuals aged 18-20 days. Larger 0-2-day old fly populations were then established and fed either melatonin or vehicle as above for 18 days, after which the Malpighian tubules were micro-dissected from 50 female flies in phosphate buffered saline (PBS). Cysts were counted separately for the anterior and posterior tubules, differentiating each tubular region (i.e., proximal, intermediate and terminal), due to their known physiological differences ( [157]; reviewed in [27]). Wild-type tubules are elongated and regularly shaped, while BicC mutant tubules appear larger and deformed by cysts. Cysts were scored as any tubular deformation creating uni-or bi-lateral expansions or extra-branches as in [30]. To determine that flies ingested melatonin, green dye was added to the food and melatonin mixture. After three days, the dye can be visualized through the semi-transparent abdominal cuticle ( Figure S1). Data were plotted using the Graphpad Prism 8.0 software (https://www.graphpad.com/scientific-software/prism/) as nested distributions and analyzed statistically. Unpaired t-tests were performed with both Excel and Graphpad Prism 8.0 and the Welch's correction added, because the populations may not have equal standard deviations. p values of less than 0.05 were considered significant and indicative of cyst-reducing activity. The cystic index raw data can be found in Table S1.

Microscopy
Malpighian tubules from aged and treated flies as indicated were manually micro-dissected in 1× PBS, washed and equilibrated into a 3:1 1× PBS:glycerol solution as in [30] and photographed on a Leica MZ FLIII Fluorescence Stereomicroscope with Leica MZ series 10×/21B Widefield adjustable eyepieces equipped with a Canon DS126201 EOS 5D MARK II camera, using visible light. Canon raw files (CR2) were converted into TIF format using the Adobe Lightroom 3.2 software (Adobe Systems, San Jose, CA, USA).

Supplementary Materials:
The following are available online, Figure S1: Melatonin feeding control, Table S1: Cystic index raw data.  Conflicts of Interest: R.R. is the Chief Executive Officer and F.P. is the Chief Scientific Officer of BH BioScience. C.M.-B. and C.G. report no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.