A Personalized Model of COQ2 Nephropathy Rescued by the Wild-Type COQ2 Allele Or Dietary Coenzyme Q10 Supplementation

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A Personalized Model of COQ2 Nephropathy Rescued by the Wild-Type COQ2 Allele or Dietary Coenzyme

Q10 Supplementation

† ‡ Jun-yi Zhu,* Yulong Fu,* Adam Richman,* Zhanzheng Zhao, Patricio E. Ray, § and Zhe Han*§

Centers for *Cancer and Immunology Research and ‡Genetic Medicine Research, Children’s National Health System, Washington, DC; †Department of Nephrology, First Afﬁliated Hospital of Zhengzhou University, Zhengzhou, China; and §Department of Pediatrics, The George Washington University School of Medicine and Health Sciences, Washington, DC

ABSTRACT Clinical studies have identiﬁed patients with nephrotic syndrome caused by mutations in genes involved in the biosynthesis of coenzyme Q10 (CoQ10), a lipid component of the mitochondrial electron transport chain and an important antioxidant. However, the cellular mechanisms through which these mutations induce podocyte injury remain obscure. Here, we exploited the striking similarities between Drosophila nephrocytes and human podocytes to develop a Drosophila model of these renal diseases, and performed a systematic in vivo analysis assessing the role of CoQ10 pathway genes in renal function.

Nephrocyte-specific silencing of Coq2, Coq6,andCoq8, which are genes involved in the CoQ10 pathway that have been associated with genetic nephrotic syndrome in humans, induced dramatic adverse changes in these cells. In particular, silencing of Coq2 led to an abnormal localization of slit diaphragms, collapse of lacunar channels, and more dysmorphic mitochondria. In addition, Coq2-deficient nephrocytes showed elevated levels of autophagy and mitophagy, increased levels of reactive oxygen species, and increased sensitivity to oxidative stress. Dietary supplementation with CoQ10 at least partially rescued these defects. Furthermore, expressing the wild-type human COQ2 gene specifically in nephrocytes rescued the defective protein uptake, but expressing the mutant allele derived from a patient with COQ2 nephropathy did not. We conclude that transgenic Drosophila lines carrying mutations in the CoQ10 pathway genes are clinically relevant models with which to explore the pathogenesis of podocyte injury and could serve as a new platform to test novel therapeutic approaches.

J Am Soc Nephrol 28: 2607–2617, 2017. doi: https://doi.org/10.1681/ASN.2016060626

Steroid-resistant nephrotic syndrome (SRNS) is a The Drosophila pericardial nephrocyte (hereaf- major cause of end stage renal disease (ESRD).1–5 ter, nephrocyte) bears striking structural and func- The identiﬁcation of single gene mutations associ- tional similarities to the mammalian podocyte.10–12 ated with SRNS have yielded insights into pathogenic mechanisms, and evidence from multiple Received June 8, 2016. Accepted March 9, 2017. studies points to the podocyte as an important target of cellular injury.4,6–9 Increased understanding J.-y.Z. and Y.F. contributed equally to this work. of molecular and cellular processes that are affected Published online ahead of print. Publication date available at by mutations, and the development of new thera- www.jasn.org. peutic treatment approaches, will be greatly aided Correspondence: Dr. Zhe Han, Children’s Research Institute, by development of an animal model system that Children’s National Health System, 111 Michigan Avenue NW, permits in vivo experimental studies relevant for Washington, DC 20010. Email: [email protected] elucidating podocyte cell function and cytotoxicity. Copyright © 2017 by the American Society of Nephrology

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Table 1. Genes involved in CoQ biosynthesis and associated with clinical renal pathology Representative Human Gene Name Protein Function Drosophila Ortholog Conservation Score Nephropathy PDSS1 (COQ1 subunit 1) Catalytic subunit of COQ1, Qless 9 None identified synthesis of CoQ polyisoprene tail PDSS2 (COQ1 subunit 2) Regulatory subunit of COQ1 Pdss2 (CG10585) 10 NS COQ2 Transferase, links Coq2 (CG9613) 10 FSGS parahydroxybenzoate redox-active head precursor to polyisoprene tail COQ3 Methylase, modification of Coq3 (CG9249) 10 None identified CoQ redox-active head COQ4 Unknown function Coq4 (CG32174) 10 None identified (regulatory?) COQ5 Methylase, modification of Coq5 (CG2453) 9 None identified CoQ redox-active head COQ6 Hydroxylase, modification of Coq6 (CG7277) 10 SRNS CoQ redox-active head COQ7 Hydroxylase, modification of Coq7 (CG14437) 8 None identified CoQ redox-active head COQ8 (ADCK4) Kinase (regulatory?) Coq8/Adck4 (CG32649) 9 SRNS COQ9 Unknown function (regulatory?) Coq9 (CG30493) 10 Renal tubulopathy Human COQ genes and Drosophila orthologs are shown, with degree of homology (conservation score: 1–10 [lowest to highest] scale).34 Representative mani- festations of kidney disease are indicated for mutation of PDSS2,16 COQ2,17 COQ6,9 COQ8,4 and COQ9.35

Combining this feature with the extensive genetic resources RESULTS available in Drosophila for conditionally manipulating gene expression in specific cells and tissues at distinct stages, we Silencing CoQ10 Biosynthesis Pathway Genes in have developed the fly as a model system to investigate Nephrocytes genetic, molecular, and cellular targets of cell injury in The CoQ10 synthesis pathway genes are highly conserved from nephrocytes that are relevant to podocytes, thereby con- Drosophila to humans. In humans, the genes include PDSS1 tributing to understanding of renal cell injury and kidney (COQ1 subunit 1), PDSS2 (COQ1 subunit 2), COQ2, COQ3, disease processes.4,5,13–15 COQ4, COQ5, COQ6, COQ7, COQ8,andCOQ9.Theirfunc- Coenzyme Q10 (CoQ10)deficiencies have been linked to tions are listed in Table 1. In Drosophila, the homologs of severe renal pathologies including SRNS, collapsing glo- PDSS1 and COQ2 are named Qless and Coq2,respectively. 4,6,9,16,17 merulopathy, and tubular interstitial diseases. Here, we have named the remaining Drosophila CoQ10 syn- Among genes encoding enzymes involved in the biosynthe- thesis pathway genes Pdss2, Coq3, Coq4, Coq5, Coq6, Coq7, sis of CoQ10, mutations in PDSS2, COQ2, COQ6, COQ8, Coq8,andCoq9, corresponding to their human homologs and COQ9 have been linked to renal pathologies.4,6,9,16 (Table 1). The human genes that have been associated with CoQ10 plays an essential role in the mitochondrial respira- the development of nephrotic syndrome (NS), focal glomer- tory chain and protects against damage from reactive oxy- ulosclerosis (FSGS), SRNS, and renal tubulopathy are indi- 6,18,19 gen species (ROS). The precise mechanism of CoQ10 cated in Table 1. We generated Drosophila transgenic lines in deficiency induced cellular pathology is not defined, but which specific Coq gene expression was silenced in nephro- occurs in the context of abnormal mitochondrial function and cytes. This strategy used the UAS-Gal4 system21 in which flies increased ROS formation.18 Patients carrying mutations result- carried a Dot-Gal4 driver construct whereby the Dot gene en- 22 fi 5,13–15 ing in primary CoQ10 deficiency present with very heterogeneous hancer directed nephrocyte-speci c expression of the symptoms.19 It remains unclear why some patients manifest yeast Gal4 transcription factor, which in turn promoted ex- with severe renal disease. CoQ10 deficiency is unique among pression of a UAS-Coq-RNAi transgene that silenced the en- mitochondrial disorders in that early supplementation with dogenous Coq target gene. 9,20 CoQ10 can prevent the onset of disease. Here, we utilized Drosophila as an in vivo model system to Coq Gene Silencing Affected Nephrocyte Cell Function investigate the phenotypes associated with CoQ10 deficiency The principal functions of the nephrocyte are first, to filter induced by the systematic silencing of all of the fly Coq genes hemolymph, and second, to take up filtered low molecular specifically in nephrocytes. Our findings validate the clinical weight proteins and toxins for recycling and sequestration, relevance of our experimental model system to study the path- respectively.11,12 We took advantage of the second role to de- ogenesis of CoQ10-related human renal diseases. velop in vivo quantitative assays to measure the effects of gene

2608 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 2607–2617, 2017 www.jasn.org BASIC RESEARCH silencing on nephrocyte uptake functions. We examined the ability of nephrocytes expressing Coq gene silencing RNAi transgenes to take up a fluorescent hemolymph marker protein. In this assay, flies carried a MHC-ANF-RFP transgene, in which a myosin heavy chain (MHC) promoter drove expression of an atrial natriuretic peptide- red fluorescent protein (ANF-RFP) fusion protein. Muscle cells secreted ANF-RFP into the fly hemolymph from which it was filtered and endocytosed by nephrocytes, and intracellular fluorescence was detected and quantitated.14 To confirm nephrocyte cell identity, flies also carried a green fluorescent protein (GFP) transgene expressed under the control of the Drosophila Hand gene promoter (Hand-GFP).23 MHC-ANF-RFP and Hand-GFP transgenes were combined in flies carrying Dot-Gal4 driver22 and UAS-Coq-RNAi constructs silencing the endogenous Coq Figure 1. Hemolymph protein marker ANF-RFP and AgNO levels in nephrocytes target genes in nephrocytes. 3 expressing Coq-RNAi transgenes. (A) Fluorescence micrographs showing nephrocytes We systematically examined the effects of adult flies 1-day postemergence. ANF-RFP fluorescence (red) is shown in the left on ANF-RFP fusion protein uptake of panels. Right panels show RFP (red) merged with GFP (green, mostly nuclear). A GFP fi nephrocyte-speci c silencing of Coq genes transgene is expressed under the control of a Hand gene enhancer (Hand-GFP)to 1–9 and Pdss2.AsshowninFigure1A,control confirm pericardial nephrocyte cell identity.23 All flies are transgenic for Hand-GFP. nephrocytes of adult flies not expressing a Control flies carry the Dot-Gal4 driver but no RNAi construct. Coq-IR flies carry Dot- Coq-RNAi transgene contained readily de- Gal4 driving an RNAi transgene to silence expression of Coq2, Coq6,orCoq8 genes. tectable levels of ANF-RFP. We observed (B) Quantification of nephrocyte RFP fluorescence, expressed relative to control value. that silencing of Coq2, Coq6,andCoq8 For each genotype, 30 nephrocytes (six nephrocytes from each of five flies) were , (but no other Coq genes) led to signifi- examined (*P 0.05). (C) Photomicrographs showing nephrocytes of third instar larvae fl fl cantly reduced levels of intracellular reared on standard y food supplemented with AgNO3. Control ies carry the Dot- Gal4 driver but no RNAi construct. Coq-IR flies carry Dot-Gal4 driving an RNAi ANF-RFP marker protein relative to con- transgene to silence expression of the indicated Coq gene. Scale bar, 20 microns. (D) trol nephrocytes (Figure 1B and Supple- Quantification of AgNO3, expressed relative to control value. For each genotype, 30 mental Figure S1). nephrocytes (six nephrocytes from each of five larvae) were examined (*P,0.05). Asecondin vivo nephrocyte functional assay was used to analyze the effects of Coq gene silencing. For these studies, toxic silver nitrate (AgNO3) was added and slit diaphragms are regularly spaced along the circumfer- to the standard diet of developing larvae. Ingested AgNO3 was taken ence of the cell. A single slit diaphragm is located at the up by normal nephrocytes and sequestered intracellularly (Figure “mouth” of each channel. Silencing of Coq2 expression led 1C, Control), thereby protecting the animal from systemic expo- to striking disruption of normal channel spacing and mor- sure to the toxin.11 We observed that RNAi mediated silencing of phology and slit diaphragm localization (Figure 2C). Regular Coq2, Coq6,andCoq8 gene expression led to decreased accumula- spacing was interrupted (Figure 2D), and some channels were tion of AgNO3 in nephrocytes (Figure 1, C and D), a phenotype dramatically narrowed and elongated. Mislocalized “ectopic” that was not observed upon silencing any other Coq genes. slit diaphragms were observed, frequently forming ladder-like arrays along a single channel (Figure 2, B and E). Quantitative Coq2 Gene Silencing Induced Abnormalities in Slit analysis indicated that slit diaphragm mislocalization was not Diaphragm and Lacunar Channel Ultrastructure associated with an increased number of total slit diaphragms We used transmission electron microscopy (TEM) to deter- (Figure 2F). mine if defects in uptake and accumulation of ANF-RFP and AgNO3 because of Coq2 gene silencing were associated with Coq2 Gene Silencing Led to Increased Numbers of abnormalities in the characteristic nephrocyte slit diaphragm Dysmorphic Mitochondria 10 and lacunar channel ultrastructure. As shown in Figure 2, A Because CoQ10 (ubiquinone) is a required component of the and B, in normal (Control) nephrocytes the lacunar channels mitochondrial electron transport chain, we examined

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mitochondrial morphology by TEM analysis in nephrocytes expressing the Coq2 gene silencing transgene. Coq2 gene silencing led to increased numbers of mitochondria (Figure 3, A and B), essentially all of which displayed aberrant morphology, characterized by dramatic reduction of inner mitochondrial membranes and shortened cristae (Figure 3, A and C).

Coq2 Gene Silencing Led to Increased Autophagy and Mitophagy Mitochondrial structural impairment plays a pathogenic role in various kidney diseases and therefore the removal of damaged mitochondria is fundamental for renal health.24 Our observation that Coq2 gene silencing in nephrocytes greatly increased numbers of dysmorphic mitochondria led us to speculate whether cellular responses aimed at removing damaged mitochondria might be upregulated. Autophagy, and more specifically mitophagy, is an important mechanism of mitochondrial quality control.24 TEM analysis revealed that nephrocyte autophagosome density was significantly increased as a result of Coq2 gene silencing (Figure 3, D and E). Tofurther examine the extent to which autophagy and specifically mitophagy might be activated as a result of Coq2 gene silencing, we used a strategy involving nephrocyte expression of a transgenic marker-protein Atg8 that is doubly fluorescence-labeled with GFP and mCherry (GFP-Atg8-mCherry).25 This Atg8 marker is present in autophagosomes and accumulates in autolysosomes. In the acidic lysosome lumen the GFP fluores- Figure 2. Coq2 gene silencing induced abnormal slit diaphragm localization and col- cence undergoes rapid quenching, whereas lapsed lacunar channels with multiple slit diaphragms (A) TEM showing normal (control) fl nephrocyte ultrastructure with slit diaphragms and lacunar channels uniformly spaced mCherry retains uorescence activity. Thus, along the circumference of the cell. Slit diaphragms localized exclusively at the mouth of only a very minor subset of autolysosomes, the channel. In Coq2-IR nephrocytes, channels appeared narrower (collapsed) and in- if any, will exhibit GFP fluorescence. Be- terchannel spacing was interrupted and irregular. Slit diaphragms occurred not only at the cause Atg8 accumulates to high levels in channel mouth but also ectopically along the interior channel membranes. Scale bar, autolysosomes, mCherry fluorescence is 200 nm. (B) Higher magnification TEM comparing normal control and Coq2-IR slit di- relatively stronger in this compartment aphragm and lacunar channel ultrastructure. Ectopic slit diaphragms arranged in ladder- than in autophagosomes. like configuration at points of channel narrowing are indicated by arrows. Scale bar, 100 As shown in Figure 4A, in control nm. (C) Quantitation of normally localized slit diaphragms in control versus Coq2-IR nephrocytes (upper three panels) express- nephrocytes. Average number of slit diaphragms positioned at mouths of lacunar channels ing GFP-Atg8-mCherry neither GFP per 2000 nm of cell circumference (*P,0.05). (D) The average distance (in nm) between nor mCherry fluorescence was detectable normally localized slit diaphragms in control versus Coq2-IR nephrocytes (*P,0.05). (E) Quantitation of ectopic slit diaphragms in control versus Coq2-IR nephrocytes. Average in a punctate pattern indicative of Atg8 in number of slit diaphragms positioned along interior channel membranes per 2000 nm of either autophagosomes or autolysosomes. cell circumference (*P,0.05). (F) Quantitation of slit diaphragms (normally localized plus Silencing of Coq2 in nephrocytes (Figure ectopic) in control versus Coq2-IR nephrocytes. Total number of slit diaphragms per 2000 4A, lower three panels) was associated nm of cell circumference (*P,0.05). with low GFP and abundant mCherry

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observed by TEM (Figure 3). We again observed very signiﬁcant upregulation of Atg8 (Figure4,B,D,andE).Mergedimages showed overlap of mitochondrial GFP plus Atg8-RFP induced by Coq2 gene silencing (Figure 4, B and E), indicating the presence of mitochondria in autophagosomes and thus revealing the occurrence of mitophagy. It was not clear from this analysis what percentage of nephrocyte mitochondria under- went degradation in autolysosomes.

Dual Knockdown of Coq2 and Autophagy Genes in Nephrocytes Induces Synergistic Effects on Protein Uptake and Intracellular ROS Levels In order to ascertain whether Coq deficiency and autophagy pathways intersect in nephrocytes, we analyzed the effects of combined silencing of Coq2 and either of the autophagy genes Atg-127 or Atg-6.28 As shown in Figure 5, A and B, the silencing of Atg-6 alone markedly reduced RFP levels but Atg-1 silencing had no effect. Simulta- neously silencing Coq2 and either Atg-1 or Atg-6 led to a more severe RFP phenotype Figure 3. Coq2 gene silencing induced elevated numbers of autophagosomes and than either Coq2 or Atg gene alone, suggest- fi abnormal mitochondria in nephrocytes. (A) Upper panels: TEM showing normal mito- ing that Coq de ciency and autophagy chondria (m) in control nephrocyte of a wild-type fly. In Coq2-IR nephrocytes the mito- pathways are linked. chondria are more abundant and morphologically abnormal. Autophagosomes (ap) are ROS production and oxidative stress in evidence. Scale bar, 300 nm. Lower panels: higher magnification electron micrographs have been implicated in renal disease as- 18 comparing normal control and Coq2-IR nephrocyte mitochondria. In nephrocytes in sociated with CoQ10 deficiency. Conse- which Coq2 expression was silenced the mitochondria exhibited shortened cristae and quently, we examined the effect of Coq2 fewer inner mitochondrial membranes. Scale bar, 200 nm. (B) Quantitation of mito- gene silencing on ROS levels in nephro- chondria in control versus Coq2-IR nephrocytes. Average number of mitochondria per m 2 , cytes. Dihydroethidium (DHE) was used 4 m area of cytoplasm (*P 0.05). (C) Percentage of mitochondria exhibiting abnormal 29,30 , as a redox indicator. Reduced DHE morphology in control versus Coq2-IR nephrocytes (*P 0.05). (D) TEM showing mi- fl tochondria and autophagosomes in control nephrocyte of a wild-type fly. In Coq-IR uoresces blue, but undergoes a shift to fl nephrocytes, the number of autophagosomes was increased. Scale bar, 300 nm. (E) red uorescence when oxidized, and in- Quantitation of autophagosomes in control versus Coq2-IR nephrocytes. Average tercalates into DNA. Coq2 silencing alone number of autophagosome per 4 mm2 area of cytoplasm (*P,0.05). induced an approximately three-fold increase in DHE red fluorescence intensity (Figure 5, C and D). Silencing either Atg1 fluorescence. Merged images confirmed the presence of few or Atg6 alone had no effect. Simultaneous silencing of Coq2 autophagosomes (yellow) and numerous autolysosomes and either Atg6 or Atg1 led to significantly higher DHE fluo- (red). This result indicated that Coq2 gene silencing led to rescence than Coq2 alone (Figure 5, C and D), again suggest- greatly increased autophagy. In order to investigate the effect ing linkage of Coq and autophagy pathways. of Coq2 gene silencing on mitophagy, we generated a Drosophila strain in which nephrocyte mitochondria are fluorescence- CoQ10 Supplementation Rescued Coq2 Silencing labeled with GFP and nephrocytes express an Atg8-RFP fusion Associated with Nephrocyte Protein Uptake protein.26 In nephrocytes of control flies, Atg8-RFP fluorescence Deficiency, Ultrastructural Abnormalities, and Elevated was very low and no combined/overlapping mitochondrial GFP ROS 31 plus Atg8-RFP (yellow fluorescence) was detected (Figure 4, B, We tested whether supplementing the flydietwithCoQ10 upper panels, and E). Coq2 gene silencing in nephrocytes was could rescue deficient ANF-RFP uptake and abnormal slit di- associated with a significant increase in GFP fluorescence (Fig- aphragm and lacunar channel morphology associated with ure 4, B and C) consistent with increased mitochondrial density Coq2 gene silencing (Figure 6). We observed no increase in

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ANF-RFP uptake when wild-type control ﬂies were fed a diet supplemented with 1% or 5% CoQ10. By contrast, nephrocytes expressing Coq2-RNAi (Coq2-IR)showed normal levels of ANF-RFP uptake when ﬂies were reared on a diet containing 5% CoQ10 (Figure 6, A and B). We furthermore observed that 5% CoQ10 diet supplementation largely restored normal slit diaphragm and lacunar channel morphology in nephrocytes expressing the Coq2-IR transgene (Figure 6, C, D, and E). We further observed that ROS levels could be lowered in Coq2-IR nephrocytes by administration of 5% CoQ10 (Figure 6, F and G).

Lower panels show nephrocytes in which Coq2 gene expression was silenced (Coq2-IR) in which a few autophagosomes were present (GFP, green; GFP-Atg8-mCherry, yellow) and autolysosomes were highly abundant (mCherry, red). (B) Fluorescence micrographs showing nephrocytes of adult female flies 1-day postemergence, expressing GFP-labeled mitochondria and Atg8-RFP marker protein in nephrocytes. Dotted line indicates nephrocyte cell boundary. Upper panels show control nephrocytes (normal Coq2 gene expression) with labeled mitochondria (Mito-GFP, green), essentially undetectable autophagy (Atg8-RFP, red), and no overlapping yellow fluorescence in merged images (Mito-GFP Atg8-RFP). Lower panels show nephrocytes in which Coq2 was silenced (Coq2-IR)withincreasedmi- tochondrial fluorescence (higher Mito-GFP fluorescence, green), fluorescence because of Atg8 in autophagosomes/autolysosomes (Atg8-RFP, red), and overlap of fluorescence indicating mitophagy (Mito-GFP Atg8-RFP, yellow). (C) Quantitative comparison of Mito- GFP fluorescence in control versus Coq2-IR nephrocytes, expressed relative to control. Figure 4. Coq2 gene silencing induced autophagy and mitophagy. (A) Fluorescence The increased fluorescence due to Coq2 si- micrographs showing nephrocytes of adult female flies 1-day postemergence, ex- lencing is shown relative to control. For each pressing a dual-labeled GFP-Atg8-mCherry autophagy marker protein specifically in group, 30 nephrocytes (six nephrocytes from nephrocytes (UAS-GFP-Atg8-mCherry construct driven by Dot-Gal4). Dotted lines each of five flies) were examined (*P,0.05). indicate nephrocyte cell boundary. Atg8 is present in autophagosomes and sub- (D) Quantitative comparison of Atg8-RFP fluo- sequently accumulates in autolysosomes. Fluorescence from the compartmentalized rescence in control versus Coq2-IR nephrocytes, marker protein is punctate in appearance. Both GFP and mCherry fluorescence can expressed relative to Control. For each group, be detected in autophagosomes. In the acidic lysosome lumen, GFP fluorescence 30 nephrocytes (six nephrocytes from each undergoes rapid quenching whereas mCherry retains fluorescence activity. There- of five flies) were examined (*P,0.05). (E) fore only very early autolysosomes will transiently exhibit GFP fluorescence. Be- Quantitative spatial comparison of Mito-GFP cause Atg8 accumulates to high levels in autolysosomes, mCherry fluorescence is (green), Atg8-RFP (red), and Mito-GFP Atg8-RFP relatively stronger in this compartment. Upper panels show control nephrocytes (yellow) fluorescence in control versus Coq2-IR (normal Coq2 gene expression) in which autophagy is essentially undetectable. nephrocytes.

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DISCUSSION

Here, we found that a Drosophila model of COQ2 nephropathy is a powerful in vivo system to study the molecular mechanisms underlying pathogenesis of this renal disease, as well as the roles of other CoQ10- related gene mutations in causing human nephropathies. More specifically, we found that silencing the expression of Coq2, Coq6, or Coq8 genes in nephrocytes led to significant deficits in protein uptake and toxin sequestration, two critical nephrocyte functions. In addition, the Coq2 knockdown strikingly altered the localization of slit diaphragms in nephrocytes, as well as the morphology of the lacunar channels, thus disrupting critical structures needed for hemolymph filtration and endocytosis.10 Furthermore, we showed that the phenotypes associated with Drosophila Coq2 gene silenc- Figure 5. Simultaneous silencing of Coq2 and autophagy genes induces synergistic ing could be rescued either by dietary CoQ10 effects. (A) Fluorescence micrographs showing nephrocytes of 1-day postemergence supplementation, or by the expression of adult flies transgenic for Hand-GFP (GFP expressed under the control of a Hand gene the wild-type human COQ2 gene, but not enhancer to confirm pericardial nephrocyte cell identity).23 ANF-RFP (red) is merged by expressing a mutant COQ2 allele derived with GFP (green, predominantly in nucleus). Control flies carry the Dot-Gal4 driver from a COQ2 nephropathy patient.6 Our but no RNAi construct. Gene-IR flies carry Dot-Gal4 driving RNAi transgenes findings thus demonstrate that a patient- silencing endogenous Coq2, Atg1,orAtg6 genes singly or in combination. (B) derived mutation contributing to the devel- Quantitation of nephrocyte RFP levels, relative to control. For each genotype, 30 opment of a specific glomerular disease can fi fl nephrocytes (six nephrocytes from each of ve ies) were examined (* indicates sig- be introduced into our Drosophila model, in fi # fi ni cance compared with control, indicates signi cance compared with Coq2-IR; effect creating a personalized approach to in- P,0.05). (C) Oxidized DHE fluorescence (red) in nephrocytes of third instar larvae. vestigate the pathogenesis and even treat- Dashed lines indicate cell nuclei (determined from DAPI staining, not shown). (D) fi fl ment of speci c renal diseases in vivo. Quantitation of DHE uorescence intensity expressed relative to control nephrocytes. fi For each genotype, 30 nephrocytes (six nephrocytes from each of five larvae) were ex- Advances in the identi cation of genetic amined (* indicates significance compared with control, # indicates significance com- lesions underlying the pathogenesis of pared with Coq2-IR; P,0.05). SRNS have led to significant insights into molecular and cellular mechanisms contributing to this disease. We have previously Drosophila Coq2 Gene Silencing Rescued by Normal shown that Drosophila nephrocytes can be used to model Human COQ2 Gene Expression but Not by a Patient- pathogenic processes induced by silencing genes that are af- Derived Mutant COQ2-S146N Allele fected in specific human renal diseases.4,5,15 In this study, we We tested whether the human COQ2 gene could complement have extended this approach to model human renal diseases Drosophila Coq2 gene knockdown and rescue nephrocyte func- associated with alterations of the CoQ10 synthesis pathway, tion in vivo. We cloned a wild-type COQ2 cDNA and generated a which are known to induce podocyte injury. To do so, we UAS-COQ2 transgenic fly line. When combined in a Dot-Gal4; quantitatively assessed deficits in two critical nephrocyte func- UAS-Coq2-RNAi;UAS-COQ2 genetic background, nephrocytes tions, hemolymph protein uptake and AgNO3 sequestration, accumulated ANF-RFP at significantly higher levels than ob- using well established functional assays that were previously served in Dot-Gal4;UAS-Coq2-RNAi flies, demonstrating rescue validated in our laboratory.13,14 by COQ2 (Figure 7). To further demonstrate the clinical rele- Only two genes involved in the flyCoQ10 synthesis pathway vance of the Drosophila model as a platform for studies of COQ have thus far been named (Qless and Coq2). In this study, we gene mutations underlying kidney disease, we generated a fly named the remaining fly genes after their corresponding hu- line combining Dot-Gal4;UAS-Coq2-RNAi with a patient- man homologs (Table 1). Functional deficits in nephrocyte 6 derived COQ2-S146N mutant allele. AsshowninFigure7, protein uptake and AgNO3 sequestration were observed in the COQ2-S146N allele failed to rescue the endogenous Coq2 young adult flies and larvae, respectively, after silencing of silencing–induced defect in ANF-RFP accumulation. Coq2, Coq6,andCoq8 genes but not other genes involved in

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CoQ10 biosynthesis. This observation sug- gests that Coq2, Coq6,andCoq8 may play critical roles in the CoQ10 pathway. Inter- estingly, mutations in the human COQ2, COQ6,andCOQ8 genes have been linked to the development of SRNS (Table 1). These findings may reflect similar genetic vulnerabilities of human podocytes and fly nephrocytes. CoQ10 is essential for mitochondrial electron transport. Studying nephrocytes in which Coq2 gene expression was silenced could shed light on how defective electron transport in CoQ10-deficient patients leads to abnormal podocyte structure and function. A remarkable finding from this study was that Coq2 gene silencing induced the abnormal localization of nephrocyte slit diaphragms associated with the collapse of lacunar channels. Although additional studies will be required to elucidate how Coq2 silencing causes these changes, disruption of these key nephrocyte structures is compatible with the functional deficits observed in our in vivo assays. Because such structural changes were not seen in the context of other genetic manipulations

Figure 6. CoQ10 administration rescued nephrocyte functional and ultrastructural that severely reduced nephrocyte function, defects induced by Coq2 gene silencing. (A) Fluorescence micrographs showing we speculate that lack of filtration and/or nephrocytes of 1-day postemergence adult flies. Flies were reared from embryos on uptake per se cannot be the cause of slit di- standard food supplemented with the indicated concentrations (1% or 5%) of CoQ10 aphragm mislocalization and lacunar fl (Q10). Left panels (MHC-ANF-RFP) show intracellular ANF-RFP uorescence (red). channel collapse. It is possible that in- Right panels (MHC-ANF-RFP Hand-GFP) show RFP (red) merged with GFP (green, creased levels of ROS produced by disrup- mostly nuclear). A GFP transgene is expressed under the control of a Hand gene tion of normal mitochondrial electron enhancer (Hand-GFP)toconfirm pericardial nephrocyte cell identity.23 All flies are transgenic for Hand-GFP. Control flies carry the Dot-Gal4 driver but no RNAi con- transfer reactions may injure the plasma struct. Coq2 flies carry Dot-Gal4 driving RNAi transgene silencing Coq2 expression. membrane, precipitating the rearrange- (B) Quantitation of RFP levels in control versus Coq2-IR nephrocytes (expressed rel- ment of slit diaphragms and the collapse ative to control) with no Q10,1%Q10,or5%Q10 dietary supplementation. For each of lacunar channels. Whatever the mecha- genotype, 30 nephrocytes (six nephrocytes from each of five flies) were examined nism, it is intriguing to observe that slit (*P,0.05). (C) TEM showing mislocalized slit diaphragms and irregularly spaced and diaphragms can occur at ectopic sites that collapsed lacunar channel ultrastructure induced by Coq2 silencing (upper panel) are not proximal to the basement mem- rescued by administration of 5% Q10 (lower panel). Scale bar, 300 nm. (D) Quantitation brane. Whether the ectopic slit diaphragms of ectopic slit diaphragms in Coq2-IR versus Coq2-IR plus 5% Q10 supplementation are structurally normal and functional re- nephrocytes. Average number of slit diaphragms positioned along interior channel mains to be determined. membranes per 2000 nm length of cell circumference (*P,0.05). (E) The average We observed increased ROS levels in distance (in nm) between normally localized slit diaphragms in Coq2-IR versus Coq2-IR Coq2-silenced nephrocytes. We propose plus 5% Q10 supplementation nephrocytes (*P,0.05). (F) ROS levels in normal (control) and Coq2-silenced (Coq2) nephrocytes were indicated by oxidized DHE red that nephrocytes, normally subject to ele- 32 fluorescence in the cell nucleus. ROS levels were higher in Coq2-IR nephrocytes, and vated ROS levels, are particularly suscep- feeding Coq2-IR larvae a diet supplemented with 5% Q10 reduced nephrocyte ROS tible to further increases in ROS and that levels. Dashed lines indicate cell nuclei (determined from DAPI staining, not shown). CoQ10 deficiency resulting from Coq2 gene (G) Levels of DHE red nuclear fluorescence expressed relative to normal control larval silencing pushes ROS levels above a thresh- nephrocytes fed a nonsupplemented diet. Coq2 gene silencing led to a 2.5–3-fold old of oxidative stress that results in cellular increase in ROS levels. Feeding Coq2-IR 5% Q10 lowered ROS to normal levels. In injury and loss of function. A major target fi each case, 30 nephrocytes (six nephrocytes from each of ve larvae) were examined is the mitochondrial inner membrane. In , (*P 0.05). addition, membrane lipids associated with

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autophagosomes or autolysosomes (Figure 4E), and virtually all mitochondria exhibited abnormal structures (Figure 3C). In this model, it appears that reduced CoQ10 levels resulted in damaged mitochondria (and thus compromised cell bioenergetics), triggering compensatory accumulation of abnormal mitochondria and upregulated mitophagy. Under these conditions, quality control through mitophagy was inade- quate to counter the deleterious conse- quences of mitochondrial dysfunction, including increased ROS (Figure 5). We speculate that much of the autophagy ob- Figure 7. A normal allele of the human COQ2 gene but not a mutant allele (COQ2- served in nephrocytes with silenced Coq2 S146A) rescued nephrocyte function in flies expressing Drosophila Coq2-IR.(A) Fluorescence micrographs showing uptake of ANF-RFP nephrocytes of 1-day post- expression was induced in response to emergence adult flies. Left panels (MHC-ANF-RFP) show intracellular ANF-RFP fluo- abnormal oxidative damage of cellular rescence (red). Right panels (MHC-ANF-RFP Hand-GFP) show RFP (red) merged with components, and the results of our genetic GFP (green, mostly nuclear). Hand-GFP expression confirms pericardial nephrocyte interaction experiments suggest that Coq cell identity. All flies are transgenic for Hand-GFP.Controlflies carry the Dot-Gal4 and autophagy pathways are linked. driver but no RNAi construct. Coq2-IR flies carry Dot-Gal4 driving RNAi transgene In conclusion, we have generated a new Dro- silencing the endogenous Drosophila Coq2 gene expression, and where indicated, sophila model of human COQ2 nephropathy also a UAS-COQ2 (wild-type human COQ2 allele) or a UAS-COQ2-S146N (mutant and established its clinical relevance for 6 human COQ2 allele). (B) Quantitation of RFP levels (expressed relative to control) in investigating the pathogenesis of this control versus Coq2-IR nephrocytes expressing normal COQ2 or mutant COQ2- renal disease in humans. In addition, we S146N. For each genotype, 30 nephrocytes (six nephrocytes from each of five flies) demonstrate that Drosophila nephrocytes were examined (*P,0.05). can be used to study pathogenic roles of other genes involved in the CoQ10 biosyn- the slit diaphragm are rendered susceptible to oxidative dam- thesis pathway, and as an in vivo drug-testing platform to age, affecting the dynamic actin cytoskeletal components that evaluate therapeutic approaches for treating cellular in- maintain the structural integrity of the slit diaphragm and lacunar jury and structural and functional abnormalities relevant channel network that surrounds the cell, and are necessary for the to kidney disease. essential nephrocyte functions of filtration and endocytosis of proteins and toxins. By extension to mammalian podocytes, this hypothesis provides an explanation for the susceptibility of the CONCISE METHODS kidney to injury as a result of mutations in COQ genes. Another important observation from the Drosophila model Fly Strains of human COQ2 nephropathy was increased numbers of dys- Flies were reared on standard food at room temperature or 29°C for morphic mitochondria in Coq2-silenced nephrocytes. Similar UAS-Gal4 experiments. The following strains were used in this study: findings have been reported for podocytes of patients with Hand-GFP,23 Dot-Gal4,14 MHC-ANF-RFP,14 UAS-Qless-IR, UAS- COQ2 nephropathy,6 and in a mouse kd/kd (PDSS2)disease Coq2-IR, UAS-Coq3-IR, UAS-Coq4-IR, UAS-Coq5-IR, UAS-Coq6- model.7,33 Taken together, these observations further validate IR, UAS-Coq7-IR, UAS-Coq8-IR, UAS-Coq9-IR, UAS-PDSS2-IR, the relevance of studying Drosophila nephrocytes to better un- UAS-Mito-GFP, UAS-GFP-Atg8-mCherry, UAS-Atg1-IR, UAS- derstand how CoQ10 deficiencies can affect mitochondria in ATG6-IR, UAS-Atg8-RFP (the last kindly provided by Ernst Hafen). podocytes. It is tempting to speculate that podocytes and Sources and ID numbers of silencing strains used in this study are nephrocytes normally require relatively high levels of ATP provided in Supplemental Table S1. and that critical functions of these cells are particularly sensi- tive to altered bioenergetics resulting from disrupted mito- DNA Cloning and Generation of Transgenic Fly Strains chondrial electron transfer pathways. We also observed that The wild-type COQ2 cDNA was obtained from OriGene, and encodes nephrocyte Coq2 silencing was associated with increased the common 371-aa isoform (GenBank ID: AAH08804). The mutant autophagy and mitophagy. The latter is an important mito- human COQ2-S146N cDNA6 was generated using QuikChange Site- chondrial quality control mechanism, and given the striking Directed mutagenesis kit (Agilent Technologies). To generate UAS- increase in abnormal mitochondria after Coq2 gene silencing, COQ2 and UAS-COQ2-S146N transgenic fly lines, the above cDNAs it was not surprising to observe upregulated mitophagy. How- were cloned into the pUAST vector and introduced into the germ cells of ever, 50% of mitochondria remained unassociated with flies by standard P element–mediated germline transformation.

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RFP Uptake Assay ROS Assay Flies from the MHC-ANF-RFP, Hand-GFP,andDot-Gal4 transgenic Nephrocytes dissected from third instar larvae were maintained in lines were crossed to flies from the UAS-Coq-RNAi transgenic lines at Schneider Medium (Gibco) containing 10 mM DHE (Invitrogen) for 25°C.14 One day after egg-laying, embryos were shifted to 29°C. RFP 30 minutes at room temperature. Nephrocytes were fixed and imaged uptake by pericardial nephrocytes was assessed in adult flies 1 day by fluorescence microscopy. postemergence by dissecting heart tissues into Drosophila Schneider Medium (Gibco) and examining cells by fluorescence microscopy. fi $ For quanti cation, 20 nephrocytes were analyzed from each of three ACKNOWLEDGMENTS female flies per genotype. The results are presented as mean6SD. Sta- tistical significance was defined as P,0.05. We thank the Bloomington Drosophila Stock Center, the Vienna Drosophila Resource Center, and Ernst Hafen for flystocks.Z.H. AgNO3 Uptake Assay was supported by National Institutes of Health (NIH) R01 grant Flies of the appropriate genotype were allowed to lay eggs on standard DK098410. P.E.R. was supported by NIH R01 grants DK49419, apple juice agar plates for 24 hours. Freshly emerged first instar larvae DK103564, and DK108368. were transferred to agar-only plates supplemented with regular yeast paste containing AgNO3 (2.0 g yeast in 3.5 ml 0.0005% AgNO3 so- 14 lution) and allowed to develop at 29°C until adulthood. AgNO3 DISCLOSURES uptake by pericardial nephrocytes was assessed in adult flies 1-day None. postemergence by dissecting heart tissues into Drosophila Schneider Medium (Gibco) and examining cells by phase-contrast microscopy. fi $ For quanti cation, 20 nephrocytes were analyzed from each of REFERENCES three female flies per genotype. The results are presented as mean6 SD. Statistical significance was defined as P,0.05. 1. 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