Cyst Reduction in a Polycystic Kidney Disease Drosophila Model Using Smac Mimics

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited malady affecting 12.5 million people worldwide. Therapeutic options to treat PKD are limited, due in part to lack of precise knowledge of underlying pathological mechanisms. Mimics of the second mitochondria-derived activator of caspases (Smac) have exhibited activity as antineoplastic agents and reported recently to ameliorate cysts in a murine ADPKD model, possibly by differentially targeting cystic cells and sparing the surrounding tissue. A first-in-kind Drosophila PKD model has now been employed to probe further the activity of novel Smac mimics. Substantial reduction of cystic defects was observed in the Malpighian (renal) tubules of treated flies, underscoring mechanistic conservation of the cystic pathways and potential for efficient testing of drug prototypes in this PKD model. Moreover, the observed differential rescue of the anterior and posterior tubules overall, and within their physiologically diverse intermediate and terminal regions implied a nuanced response in distinct tubular regions contingent upon the structure of the Smac mimic. Knowledge gained from studying Smac mimics reveals the capacity for the Drosophila model to precisely probe PKD pharmacology highlighting the value for such critical evaluation of factors implicated in renal function and pathology.


Introduction
Autosomal dominant polycystic kidney disease (ADPKD) induces the formation of cysts along the entire renal tubule, predominantly at the terminal region and the collector tubule, as well as extra-renal manifestations [1]. Inherited through a monogenic pattern with the most frequent mutations affecting the PKD1 or PKD2 gene, ADPKD displays high heterogeneity in both phenotype and speed of progression [1]. Abnormal proliferation of the epithelial tubular cells during development gives rise to cysts prenatally. Cysts become more numerous with age, gradually enlarging and filling with fluid [1]. Animal models of PKD have been invaluable for defining disease progression and identifying key molecular alterations; however, the precise mechanisms underlying disease pathology remain to be elucidated at the molecular level [2]. The most recent addition to the arsenal of PKD animal models has been the fruit fly, Drosophila melanogaster. Mutants for the Bicaudal C (BicC) gene (hereby BicC flies) were found to recapitulate key molecular features of PKD, including myc over-expression and mechanistic target of rapamycin (mTOR) pathway activation [3]. Consistent with ADPKD, the BicC flies formed cysts along the entire length of the tubule, with higher frequency at the intermediate, terminal and (peptide 1, Figure 1) and three Smac mimics 2-4 were administered to the BicC cystic flies to reduce cyst formation.

Fly Lines and Genetics
Fly breeding and care were previously described in detail [3]. In brief, flies were grown on cornmeal agar (Jazzmix, Fisher Scientific Canada, Ottawa, ON) at 25 °C and aged as indicated. Oregon R (Ore R ) wild-type flies were maintained as in [3]. BicC mutants were generated by crossing Df(2L)RA5/CyO (BicC-encompassing deletion obtained from Bloomington Drosophila Stock Center) virgin females with one of the two hypomorphic BicC mutations, BicC YC33 /CyO and BicC IIF34 /CyO and selecting straight-winged progeny (Df(2L)RA5/BicC YC33 , BicC Δ/YC33 and Df(2L)RA5/BicC IIF34 , BicC Δ/IIF34 ). The two BicC allelic combinations produced truncated proteins and sterile BicC flies [3]. Eclosed adult flies were collected every two days to yield 0-2-day old populations and aged as described.

Cystic Index
For the cyst analysis, 0-2-days old flies were aged seven days (i.e., 7-9 days old) and were fed every three days with 2 mL of cornmeal food and equal volumes (50 µL) of either vehicle (water) or an aqueous solution containing peptide 1 or Smac mimic 2-4. Each compound was used at 20 µM and was administered for 20 days (i.e., until 27-29 days old). Malpighian tubules were microdissected from 25-50 female flies in phosphate buffered saline (PBS) and the number of cysts was scored separately in anterior and posterior tubules and assigned to each tubular region (i.e., proximal, intermediate and terminal [3,9]), as follows. At one extremity, the proximal region consists of about 15% of the tubule length, tends to have an opaque whitish content (posterior tubule), is thinner than adjacent regions (especially in the anterior tubule) and often exhibits a slight constriction terminally. The neighboring intermediate region has darker contents and consists of about 55% of the tubule length. The terminal region is often translucent, directly connected to the collecting tubule, and consists of the remaining 30% of the tubule length. Cysts were considered tubular deformations creating uni-or bi-laterally protuberant pouches. Extra-branches (as in [3]) were counted as cysts. Values were plotted using the Prism 8.0 software (Graphpad Software, San Diego, CA, USA) as nested distributions. Statistical analyses were performed as unpaired t tests with Welch's correction (the populations may not have equal standard deviations). The raw data counts are listed in supplementary Table S1.

Microscopy
Malpighian tubules from appropriately aged and treated flies were micro-dissected in 1× PBS, equilibrated into a 3:1 1× PBS:glycerol solution 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

Fly Lines and Genetics
Fly breeding and care were previously described in detail [3]. In brief, flies were grown on cornmeal agar (Jazzmix, Fisher Scientific Canada, Ottawa, ON) at 25 • C and aged as indicated. Oregon R (Ore R ) wild-type flies were maintained as in [3]. BicC mutants were generated by crossing Df(2L)RA5/CyO (BicC-encompassing deletion obtained from Bloomington Drosophila Stock Center) virgin females with one of the two hypomorphic BicC mutations, BicC YC33 /CyO and BicC IIF34 /CyO and selecting straight-winged progeny (Df(2L)RA5/BicC YC33 , BicC ∆/YC33 and Df(2L)RA5/BicC IIF34 , BicC ∆/IIF34 ). The two BicC allelic combinations produced truncated proteins and sterile BicC flies [3]. Eclosed adult flies were collected every two days to yield 0-2-day old populations and aged as described.

Cystic Index
For the cyst analysis, 0-2-days old flies were aged seven days (i.e., 7-9 days old) and were fed every three days with 2 mL of cornmeal food and equal volumes (50 µL) of either vehicle (water) or an aqueous solution containing peptide 1 or Smac mimic 2-4. Each compound was used at 20 µM and was administered for 20 days (i.e., until 27-29 days old). Malpighian tubules were micro-dissected from 25-50 female flies in phosphate buffered saline (PBS) and the number of cysts was scored separately in anterior and posterior tubules and assigned to each tubular region (i.e., proximal, intermediate and terminal [3,9]), as follows. At one extremity, the proximal region consists of about 15% of the tubule length, tends to have an opaque whitish content (posterior tubule), is thinner than adjacent regions (especially in the anterior tubule) and often exhibits a slight constriction terminally. The neighboring intermediate region has darker contents and consists of about 55% of the tubule length. The terminal region is often translucent, directly connected to the collecting tubule, and consists of the remaining 30% of the tubule length. Cysts were considered tubular deformations creating unior bi-laterally protuberant pouches. Extra-branches (as in [3]) were counted as cysts. Values were plotted using the Prism 8.0 software (Graphpad Software, San Diego, CA, USA) as nested distributions. Statistical analyses were performed as unpaired t tests with Welch's correction (the populations may not have equal standard deviations). The raw data counts are listed in Supplementary Table S1.

Microscopy
Malpighian tubules from appropriately aged and treated flies were micro-dissected in 1× PBS, equilibrated into a 3:1 1× PBS:glycerol solution 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).

General Synthetic Methods
Chemicals were used as received from commercial sources without further purification unless stated otherwise. All glassware was stored in the oven or flame-dried and let cool under an inert atmosphere prior to use. Anhydrous solvents (DCM, and DMF) were obtained by passage through solvent filtration systems (Glass-Contour, Irvine, CA, USA). Silica gel chromatography was performed using 230-400 mesh silica gel (Silicycle), and TLC was on glass-backed silica plates visualizing the developed chromatogram by UV absorbance or staining with ceric ammonium molybdate or potassium permanganate solutions. Nuclear magnetic resonance spectra ( 1 H and 13 C) were recorded on a Bruker AV 500 spectrometer and referenced to residual solvent in CD 3 OD (3.31 ppm, 49.0 ppm). Coupling constant J values and chemical shifts were measured in Hertz (Hz) and parts per million (ppm). Infrared spectra were recorded in the neat on a Perkin Elmer Spectrum One FTIR instrument and are reported in reciprocal centimeter (cm -1 ). Liquid chromatography−mass spectrometry (LC−MS) was performed on an Agilent Technologies 1200 series instrument in positive electrospray ionization (ESI)-time-of-flight (TOF) mode at the Université de Montréal Mass Spectrometry Facility. Sodium and proton adducts ([M + Na] + and [M + H] + ) were used for empirical formula confirmation. The peptide H-Ala-Val-Pro-Ile-NH 2 (1), Smac mimics 2 and 3, and aza-cyclohexylglycinyl-l-proline benzhydrylamide (8), all were synthesized according to published methods [49]. N-Boc-N-Methyl-l-alanine and diisopropyl ethyl amine (DIEA) were purchased from Aldrich or Alfa Aesar and used without further purification. Benzotriazol-1-yl-oxytripyrrolidino-phosphoniumhexafluoro-phosphate (PyBop) was purchased from GL Biochem™, recrystallized prior to use from dry CH 2 Cl 2 /Et 2 O (melting point, 156 • C), and stored in the dark.

Chemistry
Smac activity has been correlated to binding to IAP proteins and mimicked by its N-terminal four residue amide sequence (H-Ala-Val-Pro-Ile-NH 2 , 1, Figure 1) [50][51][52]. The purported turn conformation adopted about the central Val-Pro dipeptide in this sequence has led to the synthesis of various constrained analogs, exhibiting enhanced potency [12,53]. Noting the similar conformational preferences of indolizidinone amino acid and aza-amino acyl proline turn mimics [54][55][56][57], and the relative ease of synthesis of the latter, a series of aza-analogs were synthesized and certain were shown to induce cell death by a caspase-9 mediated apoptotic pathway in cancer cell cultures [49,57]. Notably, aza-methanopipecolate and aza-cyclohexylglycine analogs 2 and 3 were synthesized using pericyclic chemistry on the diazo dicarbonyl moiety of an azopeptide to examine the conformation of the Val residue in 1 [49,58,59]. Specifically, the Alder-ene reaction of cyclohexadiene and the Diels-Alder reaction of cyclopentadiene on N-(Cbz)azoglycinyl-proline benzhydrylamide 5 gave the unsaturated azapeptides 6 and 7, which were hydrogenated with concomitant removal of the benzyloxycarbonyl group, coupled to N-protected alanine, and deprotected (Scheme 1, [49]). Considering the tolerance of N-methyl-alanine for alanine in the terminal position [60], a similar approach was used to prepare N-methyl analog 4 by hydrogenation of azapeptide 7, coupling of the resulting aza-cyclohexylglycinyl-L-proline benzhydrylamide (8) to N-(Boc)-N-methylalanine using PyBop and cleavage of the Boc group with TFA in DCM. In cultured MCF7 breast cancer cells, aza-cyclohexylglycine analog 3 induced up to 60% cell death relative to vehicle [49]. Scheme 1. Synthesis of Smac mimics 2 and 4 (dienes, azo-and aza-residues in blue).

Effect of Smac Mimic Administration In Vivo
We have previously reported that flies mutant in the BicC gene recapitulate key features of PKD [3]. Considering that Smac mimics showed efficacy at reducing cysts in a rat PKD model [42], the results of Smac mimic administration were tested in the BicC cystic flies using two different allelic combinations. BicC mutant flies were obtained from genetic crosses of heterozygote parents consisting of CyO-balanced flies containing Df(2L)RA5 (∆, a BicC-encompassing deletion), and either the BicC YC33 or BicC IIF34 alleles similarly balanced with CyO. The BicC ∆/YC33 fly exhibits milder cystic defects than the BicC ∆/IIF34 fly [3]. Straight-winged mutants (BicC ∆/YC33 , BicC ∆/IIF34 ) from crosses were selected within two days of eclosion to yield pools of 0-2 days old flies and aged for seven days (7-9 days old), during which time the flies were kept well-fed by transferring to fresh vials twice ( Figure 2). Aged sibling flies were divided into fresh vials containing food spiked with either vehicle (water, 50 µL) or one of the Smac analogs 1-4 at 20 µM (50 µL). Flies were transferred into identical fresh vials every three days. After 20 days of treatment, the Malpighian tubules were micro-dissected from 27-29 day old flies and analyzed ex vivo. Per each condition, cystic deformities were scored using 25-50 female BicC ∆/IIF34 and BicC ∆/YC33 flies (i.e., 50-100 anterior and 50-100 posterior tubules). For each fly, cysts which were found in the two anterior and two posterior tubules were scored and charted as nested plots using the Prism 8.0 (Graphpad) software (Figures 3-6). Vehicle-treated flies presented several cysts in both the anterior and posterior MTs, especially in the terminal and intermediate regions, and fewer cysts in the proximal region, as reported previously [3]. Similar to PKD patients [1], a variable number of cysts was found in different individuals [3]. In general, Smac-mimics appeared to reduce tubular cysts in both BicC allelic combinations (Tables 1 and 2).  [3]. Similar to PKD patients [1], a variable number of cysts was found in different individuals [3]. In general, Smac-mimics appeared to reduce tubular cysts in both BicC allelic combinations (Tables 1,2).   Effects of Smac mimic administration were recorded as overall cyst reduction (Tables 1,2). Nested plots were used to represent individual variability in cyst number in the analyzed fly populations. Administration of peptide 1 to BicC Δ/YC33 flies (n = 50) reduced cystic deformities respectively by 44% and 20% (228 vs. 127 cysts and 276 vs. 220 cysts, p = 0.0005 and 0.0457) in the anterior and posterior tubules, respectively (Table 1, Figure 3A,B). Administration of mimic 2 to Testing protocol for Smac-mimic efficacy in cyst reduction. Ore R wild-type, BicC ∆/YC33 , and BicC ∆/IIF34 flies (0-2 days old) were placed in food-containing vials at age 0-2 days and transferred into fresh vials every three days. Once the age of 7-9 days was reached, flies were placed in vials containing one of each of the Smac-mimics or vehicle control, respectively. Cysts were scored on the micro-dissected MTs after 20 days, when flies reached age 27-29 days.  Effects of Smac mimic administration were recorded as overall cyst reduction ( Table 1, Table 2). Nested plots were used to represent individual variability in cyst number in the analyzed fly populations. Administration of peptide 1 to BicC ∆/YC33 flies (n = 50) reduced cystic deformities respectively by 44% and 20% (228 vs. 127 cysts and 276 vs. 220 cysts, p = 0.0005 and 0.0457) in the anterior and posterior tubules, respectively (Table 1, Figure 3A,B). Administration of mimic 2 to BicC ∆/YC33 flies (n = 50) reduced tubular cysts by 24% and 34% (216 vs. 165 cysts, p = 0.0324 and 244 vs. 162 cysts, p = 0.0017) in the anterior and posterior tubules, respectively (Table 1, Figure 3A,B). Administration of mimic 3 to BicC ∆/YC33 flies (n = 50) reduced cysts in the anterior and posterior tubules respectively by 21% and 34% (195 vs. 153 cysts, p = 0.1028 and 238 vs. 156 cysts, p = 0.0014, Table 1, Figure 3A,B). Finally, administration of mimic 4 to BicC ∆/YC33 flies (n = 50) reduced cysts in the anterior and posterior tubules respectively by 31% and 18% (134 vs. 93 cysts, p = 0.0553 and 152 vs. 124 cysts, p = 0.1877, Table 1, Figure 3A,B).  Table 1, Figure 3A,B). The analogs were next administered to the BicC Δ/IIF34 flies carrying the allelic combination which produced a more severe phenotype. Treatment with peptide 1 decreased cystic deformations respectively by 44% and 37% (total 357 vs. 199 cysts and 433 vs. 272 cysts, p < 0.0001 and 0.0002, respectively) in the anterior and posterior tubules of the micro-dissected BicC Δ/IIF34 flies (n = 50, Table  2, Figure 4A,B). Administration of mimic 2 to BicC Δ/IIF34 flies (n = 50) reduced cystic deformations by 39% in the anterior (233 vs. 142 cysts, p = 0.0011) and by 33% in the posterior (302 vs. 202 cysts, p = 0.0020) tubules (Table 2, Figure 4A,B). Administration of mimic 3 to BicC Δ/IIF34 flies (n = 25) had a milder and highly variable effect on the renal tubules and reduced cystic deformities by 11% (114 vs. The analogs were next administered to the BicC ∆/IIF34 flies carrying the allelic combination which produced a more severe phenotype. Treatment with peptide 1 decreased cystic deformations respectively by 44% and 37% (total 357 vs. 199 cysts and 433 vs. 272 cysts, p < 0.0001 and 0.0002, respectively) in the anterior and posterior tubules of the micro-dissected BicC ∆/IIF34 flies (n = 50, Table 2, Figure 4A,B). Administration of mimic 2 to BicC ∆/IIF34 flies (n = 50) reduced cystic deformations by 39% in the anterior (233 vs. 142 cysts, p = 0.0011) and by 33% in the posterior (302 vs. 202 cysts, p = 0.0020) tubules (Table 2, Figure 4A,B). Administration of mimic 3 to BicC ∆/IIF34 flies (n = 25) had a milder and highly variable effect on the renal tubules and reduced cystic deformities by 11% (114 vs. 102 cysts, p = 0.6317) and 21% (155 vs. 122 cysts, p = 0.2285) in the anterior and posterior tubules respectively (Table 2, Figure 4A,B), below statistical relevance thresholds. Finally, administration of mimic 4 to BicC ∆/IIF34 flies (n = 50) reduced cysts in the anterior and posterior tubules by 40% (199 vs. 119 cysts, p = 0.0005) and 38% (243 vs. 151 cysts, p = 0.0002) respectively (Table 2, Figure 4A,B). Parallel respective administration of analogs 1-4 to control, non-cystic Ore R wild type flies did not change MT morphology.  (Table 2, Figure 4A,B). Parallel respective administration of analogs 1-4 to control, non-cystic Ore R wild type flies did not change MT morphology.  (Table 3, 4), and individual variable cyst numbers similarly plotted ( Figure 5,6). Absolute numbers for the cysts in each region described below are listed in Table S2.  (Tables 3 and 4), and individual variable cyst numbers similarly plotted (Figures 5 and 6). Absolute numbers for the cysts in each region described below are listed in Table S2.         Table 3, Figure 5A). In the proximal region of the anterior tubules, two cysts were found in the control versus one cyst in the treated samples, precluding statistical analyses. In the posterior tubules, administration of peptide 1 diminished cysts in the intermediate region by 26% (total 109 vs. 80 cysts, p = 0.0449). In the terminal and proximal regions, a trend toward decreased cysts was observed, albeit without reaching a statistical threshold for significance (respectively 108 vs. 84 cysts, 22% reduction, p = 0.1405, and 59 vs. 56 cysts, 5% reduction, p = 0.7531, Table 3, Figure 5A).

Smac Mimics Differentially Affect Distinct Regions of the MTs
Administration of peptide 1 to BicC ∆/IIF34 flies reduced cysts in the anterior tubules by 40% and 48% (total 156 vs. 94 and 193 vs. 100 cysts, p = 0.0038 and p < 0.0001) in the terminal and intermediate regions, respectively (Table 4, Figure 6A). In the proximal region of the anterior tubules, a trend of cyst reduction was observed, albeit fewer cysts (eight and five in vehicle-and peptide 1-treated tubules) and high individual variability precluded a margin of confidence. In the posterior tubules, administration of peptide 1 reduced cysts in the terminal, intermediate and proximal regions by 32%, 41% and 39% (total 180 vs. 122, 167 vs. 98, 86 vs. 52 cysts, p = 0.0022, 0.0008 and 0.0150), respectively (Table 4, Figure 6A).
Upon treatment with mimic 4, cysts were lessened in the terminal and intermediate regions of the anterior tubules of BicC ∆/IIF34 flies (n = 50) by 38% and 42% (92 vs. 57 cysts, p = 0.0195 and 106 vs. 62 cysts, p = 0.0014), respectively (Table 4, Figure 6D). Precluding statistical analysis, only one cyst was detected in the proximal region of control tubules and no cysts in the treated tubules. Administration of mimic 4 reduced cysts in the terminal region of the posterior tubules by 51% (113 vs. 55 cysts, p < 0.0001). Less cysts were also scored in the intermediate and proximal regions, although values did not reach the significance threshold (respectively 22% reduction, 90 vs. 70 cysts, p = 0.1231, and 35% reduction, 40 vs. 26 cysts, p = 0.1069, Table 4, Figure 6D).
Ineffective cyst reduction could be partly due to flies refusing to ingest the Smac mimics. To assess their ingestion, the analogs were mixed with food and dye, and fed to the flies for four days. The green dye could be seen through the semi-transparent abdominal cuticle of the BicC flies for all mimics, confirming analog ingestion ( Figure S1). As a measure of hydrophilicity, the clogP values were calculated for the different Smac mimics (Figure 1) and found to be sufficiently low to be consistent with absorption: 1 (0.7), 2 (3.9), 3 (5.0), 4 (5.4). Together these results support the conclusion that the Smac analogs may have differential activities and/or processing.

Discussion
A systematic analysis of the influences of the Smac mimic H-Ala-Val-Pro-Ile-NH 2 (1) and constrained analogs 2-4 on renal cystogenesis has been performed using the novel BicC fly model to recapitulate features of PKD [3]. Two allelic combinations for BicC were used that yield cystic phenotypes of different severity, namely BicC ∆/YC33 and BicC ∆/IIF34 . Previously, the Smac mimic GT13072 reduced cystogenesis in a rat ADPKD model [42]. In the BicC flies, Smac mimics 1-4 ameliorated similarly the cystic condition with the strongest effects displayed in the more severely affected BicC ∆/IIF34 genotype. Peptide 1 exhibited the highest overall efficacy reducing cyst occurrence by 20-44% across genotypes at the anterior and posterior tubules. Moreover, aza-methanopipecolate 2 caused a 24-39% reduction of cysts. Aza-cyclohexylglycine 3 displayed least efficacy, but still improved significantly the posterior tubules of the BicC ∆/YC33 flies. The related N-methyl-alaninyl-aza-cyclohexylglycine analog 4 exhibited differential activity in the two BicC mutants, reducing cysts by~40% in both tubules of the BicC ∆/IIF34 flies. In contrast, analog 4 only showed a trend towards reducing cysts in the anterior tubule of the BicC ∆/YC33 flies close to the significance threshold (p = 0.0553). The Smac mimics tested appeared to differentially affect the anterior and posterior tubule pairs overall, consistent with the report of the latter having distinct transcriptomes [10] and thus different physiological specialization. Mimics 1, 2 and 3 were found to induce death of 20% (1, 2) and 60% (3) of cultured MCF7 adenocarcinoma cells [49]. Their efficacy in other PKD models and patients is unknown. Smac mimics function in context-dependent ways, likely through different IAPs to affect apoptosis via several mechanisms [12]. Due to its methyl group, N-methyl-alaninyl-aza-cyclohexylglycine analog 4 was expected to be more stable than aza-cyclohexylglycine 3 in vivo [61][62][63], which was consistent with the observed higher cyst-reducing activity.
The tubular sections responded differentially to treatment with the Smac mimics. The terminal region consistently exhibited better improvement, especially in the weaker BicC ∆/YC33 allelic combination. The flies were shown to ingest the Smac mimics. The clogP values of mimics 1-4 were also within the range consistent with effective absorption. The distinct pharmacological responses observed at different regions of the MTs may be related to variability in absorption, metabolism and response to Smac mimics within cystic cells in such regions. Regional specialization of fly MTs has been observed despite the tubular epithelium being composed by only two major cell types (reviewed in [9,64]). Cystogenesis may perturb cells and reduce the threshold for initiating cell death pathways either through caspase-dependent apoptosis or the TNF signaling pathway. The contribution of apoptosis to the early phases of ADPKD is a matter of debate [65]; however, the TNF pathway has been implicated in ADPKD-type renal cystogenesis and suggested to be the primary target of Smac mimics in a rat model of Pkd1-dependent ADPKD [42]. The results presented here predict that the BicC mutation may feature dysregulated TNF signaling in the epithelial cells of the MTs, as observed in ADPKD-type cystogenesis. The expression of TNF (Eiger) and TNF pathway components in the MT and their respective contributions to tubular function are however unknown. The human BicC orthologue BICC1 has been found to be genetically downstream of the main PKD1 gene [3]. The pharmacological response to Smac mimics was herein demonstrated to be conserved in the fly illustrating further the phenotypic and molecular similarities between PKD1-induced and BicC-induced renal cystogenesis.
Notably, pharmacological binding sites have been found to be conserved in Drosophila [66]. Chemical probing in the fly in vivo may thus rapidly pinpoint the involvement of specific pathways with complementarity to genetic analyses, indicate conserved biological activity of drug-prototypes, and provide a rapid read-out for effectiveness of pharmacological modulation of specific pathways involved in cystic pathogenesis. The Smac mimics affected different tubular regions differentially. Considering that such specificity may be conserved to humans, the development of personalized pharmacological treatments for cystic renal diseases such as PKD will benefit from precise knowledge of the cyst-ameliorating potential of different Smac mimics.