Mutations in NUP160 Are Implicated in Steroid-Resistant Nephrotic Syndrome

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Feng Zhao,1,2,3,4 Jun-yi Zhu ,2 Adam Richman,2 Yulong Fu,2 Wen Huang,2 Nan Chen,5 Xiaoxia Pan,5 Cuili Yi,1 Xiaohua Ding,1 Si Wang,1 Ping Wang,1 Xiaojing Nie,1,3,4 Jun Huang,1,3,4 Yonghui Yang,1,3,4 Zihua Yu ,1,3,4 and Zhe Han2,6

1Department of Pediatrics, Fuzhou Dongfang Hospital, Fujian, People’s Republic of China; 2Center for Genetic Medicine Research, Children’s National Health System, Washington, DC; 3Department of Pediatrics, Afﬁliated Dongfang Hospital, Xiamen University, Fujian, People’s Republic of China; 4Department of Pediatrics, Fuzhou Clinical Medical College, Fujian Medical University, Fujian, People’s Republic of China; 5Department of Nephrology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, People’s Republic of China; and 6Department of Genomics and Precision Medicine, The George Washington University School of Medicine and Health Sciences, Washington, DC

ABSTRACT Background Studies have identified mutations in .50 genes that can lead to monogenic steroid-resistant nephrotic syndrome (SRNS). The NUP160 gene, which encodes one of the protein components of the nuclear pore complex nucleoporin 160 kD (Nup160), is expressed in both human and mouse kidney cells. Knockdown of NUP160 impairs mouse podocytes in cell culture. Recently, siblings with SRNS and proteinuria in a nonconsanguineous family were found to carry compound-heterozygous mutations in NUP160. Methods We identified NUP160 mutations by whole-exome and Sanger sequencing of genomic DNA from a young girl with familial SRNS and FSGS who did not carry mutations in other genes known to be associated with SRNS. We performed in vivo functional validation studies on the NUP160 mutations using a Drosophila model. R11733 E803K Results We identified two compound-heterozygous NUP160 mutations, NUP160 and NUP160 . We showed that silencing of Drosophila NUP160 specifically in nephrocytes (fly renal cells) led to functional abnormalities, reduced cell size and nuclear volume, and disorganized nuclear membrane structure. These defects were completely rescued by expression of the wild-type human NUP160 gene in nephrocytes. R11733 By contrast, expression of the NUP160 mutant allele NUP160 completely failed to rescue nephrocyte E803K phenotypes, and mutant allele NUP160 rescued only nuclear pore complex and nuclear lamin localization defects. Conclusions Mutations in NUP160 are implicated in SRNS. Our findings indicate that NUP160 should be included in the SRNS diagnostic gene panel to identify additional patients with SRNS and homozygous or compound-heterozygous NUP160 mutations and further strengthen the evidence that NUP160 muta- tionscancauseSRNS.

J Am Soc Nephrol 30: ccc–ccc, 2019. doi: https://doi.org/10.1681/ASN.2018080786

Received August 1, 2018. Accepted February 2, 2019. Research, Children's National Health System, 111 Michigan Ave NW, Washington DC 20010, or Dr. Zihua Yu, Department of F.Z. and J.-y.Z. contributed equally to this work. Pediatrics, Fuzhou Dongfang Hospital, 156 Xi Er Huan Bei Lu, Fuzhou, Fujian 350025, China. Email: zhan@childrensnational. Published online ahead of print. Publication date available at org or [email protected] www.jasn.org. Copyright © 2019 by the American Society of Nephrology Correspondence: Dr. Zhe Han, Center for Genetic Medicine

J Am Soc Nephrol 30: ccc–ccc, 2019 ISSN : 1046-6673/3005-ccc 1 BASIC RESEARCH www.jasn.org

Nephrotic syndrome is a renal disease caused by disruption of Significance Statement the glomerular filtration barrier, resulting in massive proteinuria, hypoalbuminemia, hyperlipidemia, and edema.1 With Mutations in .50 genes can lead to monogenic steroid-resistant ne- respect to responsiveness to standard steroid therapy, nephrotic syndrome (SRNS). The authors found that a young patient with phrotic syndrome is classified into steroid-sensitive nephrotic familial SRNS and FSGS carried novel compound-heterozygous mutations in NUP160; this gene encodes nucleoporin 160 kD, one of the syndrome and steroid-resistant nephrotic syndrome (SRNS). protein components of the nuclear pore complex. Using an in vivo renal SRNS can have either an immunologic or genetic etiology.2 cell assay on the basis of Drosophila nephrocytes (an experimental The contributions of genetic factors are increasingly empha- podocyte model previously used to validate candidate renal disease sized in the growing understanding of SRNS pathogenesis. To genes and specific patient-derived mutant alleles), they validated the NUP160 date, .50 monogenic genes have been identified that cause gene variants as factors implicated in kidney pathology. The findings indicate that NUP160 should be included in the SRNS di- 3 SRNS when mutated. These include genes encoding compo- agnostic gene panel to identify additional patients with SRNS carrying nents of the slit diaphragm, such as NPHS1, NPHS2,and homozygous or compound-heterozygous NUP160 mutations. CD2AP; genes encoding actin cytoskeleton proteins, such as ACTN4, INF2,andMYO1E; genes encoding actin-regulating NUP160 small GTPases of the Rho/Rac/Cdc42 family, including transgene but not by either patient-derived mutant in vivo NUP160 ARHGDIA, ARHGAP24,andKANK; and genes encoding allele, providing evidence to implicate these nucleoporins (Nups), including NUP93, NUP107,and mutations as pathogenic. NUP205. Mutations in NPHS1, NPHS2,andCD2AP disrupt the integrity of the slit diaphragm and lead to congenital nephrotic syndrome, early-onset autosomal recessive SRNS, METHODS and early-onset FSGS, respectively.4–6 Mutations in ACTN4 change the cytoskeletal dynamics of podocytes and lead Study Participants to adult-onset autosomal dominant FSGS.7 Mutations in Written informed consent from index family members and ARHGDIA alter podocyte migration capabilities and lead to control subjects was obtained under a protocol approved by early-onset SRNS.8 Mutations in NUP93 inhibit podocyte the institutional review boards of Shanghai Ruijin Hospital and proliferation, promote podocyte apoptosis, and lead to early Fuzhou Dongfang Hospital of China. DNA samples from 520 childhood-onset SRNS.9 Mutations in NUP107 cause hypo- healthy persons were used as controls. The nonconsanguineous plastic glomerular structures and abnormal podocyte foot Chinese index family included the proband, unaffected parents, processes and lead to early childhood-onset SRNS.10 Recently, and five siblings, two of whom died from SRNS in the 1990s Braun et al.11 described mutations in NUP107, NUP85, (Figure 1C, Supplemental Table 1). DNA samples of the index NUP133,andNUP160 in 13 families with SRNS. family were available from the proband (II6), a healthy sibling The NUP160 gene (mapping to chromosome 11p) encodes (II5), and both parents (I1 and I2). Nephrotic syndrome was Nup160, which is a component of the Nup107–160 complex diagnosed on the basis of urinary protein excretion .50 mg/kg required for early stages of nuclear pore complex (NPC) as- per day with hypoalbuminemia ,25 g/L. Steroid resistance was sembly.12,13 It is expressed in both human and mouse kidney defined as a failure of induction of complete remission after cells. Knockdown of the NUP160 gene damages mouse 4 weeks of standard therapy with prednisone (2 mg/kg per day podocytes cultured in vitro.14 Within a nonconsanguineous giveninthreedivideddoses;maximum60mg/d).ESRDwas 2 Chinese family two siblings, a brother with SRNS and a sister defined as a GFR,10 ml/min per 1.73 m or the necessity of E803K with proteinuria, were both found to carry NUP160 and any RRT. Tissue biopsies were evaluated by renal pathologists. R9103 NUP160 compound-heterozygous mutations.11 We excluded the possibility that the proband carried mutations We now describe two compound-heterozygous mutations in known genes associated with SRNS (Supplemental Table 2). R11733 E803K in NUP160, NUP160 and NUP160 , identified in a young girl with familial SRNS and FSGS. This patient did not Whole-Exome Sequencing carry mutations in genes previously associated with SRNS. Genomic DNAwas purified from blood samples collected from Furthermore, we functionally validated the NUP160 muta- the proband (II6), her surviving sister (II5), and her parents tions in vivo using a Drosophila model.15–21 The Drosophila (I1 and I2) using the DNeasy Blood & Tissue Kit (Qiagen) nephrocyte system has been previously proven to be very use- using standard procedures (Figure 1C). Whole-exome capture ful for validating candidate gene mutations for involvement in and sequencing were performed on a SureSelect platform monogenic SRNS and investigating molecular disease mech- (Agilent) with 3 mg of genomic DNA from each individual anisms underlying podocyte cellular pathologies.22–25 We first using SureSelect Exome Capture System V4 (5M). The resulting showed that nephrocyte-specific silencing of the conserved libraries were sequenced on a HiSeq 2000 Multiplexed Sequenc- endogenous fly Nup160 gene induced severe renal cell defects, ing platform (Illumina) according to the manufacturer’s instruc- thereby experimentally validating NUP160 as a novel candi- tions for paired end 100-bp reads. Reads were aligned to the date renal disease gene. We then showed complete rescue of human genome reference sequence (GRCh37/hg19; build of nephrocyte phenotypes by expression of a wild-type human the UCSC Genome Browser; http://genome.ucsc.edu) using

2 Journal of the American Society of Nephrology J Am Soc Nephrol 30: ccc–ccc,2019 www.jasn.org BASIC RESEARCH

A ATG TGA

1 243 576108 9 11 12 13 14 15 16 17 1819 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

5,476 bp 196 bp

B 803 1173

Nup160

1,436 aa

pfam11715: nuleoporin Nup120/160 c.2407G>A(h) c.3517C>T(h) p.Glu803Lys p.Arg1173X

low complexity region D RefSeq

phosphorylation

II6 (P) coiled coil

C I1(F)

I2(M)

123456

II5(S)

E p.Glu803Lys p.Arg1173X

H. sapiens ESNL QHLLLSEV TDS D G ECTAAPTNR-QIIL E EL ED M. musculus ESNL QHLLLSEV TDS D G ECTAAPTNR - Q I E ILE L ED G. gallus ESNL QHLLLASEV DT D G E C A AVP TTR- Q IIE L E L ED X. tropicalis ESNL QHLLLS V E SDS F E G AL CCK I R L Q Q GGEEAAQ A D. rerio N A D L QHLLLSEV SDS D G E FA SE P V N Q - Q D II L E L K D C. intestinalis EHTL QSLGS V D NKSS E G E A L P V E N S Q RK I R L V E F DQ D. melanogaster E A S I QRLALFSQR S G KDEDCKPE R G Q E- VVV L E AL D

Figure 1. Compound-heterozygous mutations in NUP160, NUP160Glu803Lys and NUP160Arg1173X, were identiﬁed in an autosomal recessive family with steroid-resistant nephrotic syndrome. (A) Human NUP160 cDNA (NM_015231.2; 5476 bp) showing exons (numbered; alternating black and white), positions of start (ATG) and stop (TGA) codons, and mutation sites (arrows). (B) Human Nup160 protein (NP_056046.1; 1436 amino acids) structural domains, phosphorylation sites, and locations of missense (amino acid 803) and truncation (amino acid 1173) lesions. (C) A pedigree of the affected family (family members diagnosed with kidney disease are indicated in black). The arrow indicates the proband. (D) Compound-heterozygous missense and truncating NUP160 mutations identiﬁed from proband (II6; patient [P]) sequencing. Nucleotide and corresponding amino acid sequences are shown for wild-type (upper panel; in black) and mutant (lower panel; in green) alleles. Codons are underlined (green), and altered

J Am Soc Nephrol 30: ccc–ccc, 2019 NUP160 Mutations in SRNS 3 BASIC RESEARCH www.jasn.org the Burrows–Wheeler Alignment (version 0.5.9), and possible from the BDSC or the Vienna Drosophila Resource Center. duplicate paired end reads were marked using Picard version The Hand-GFP26 and MHC-ANF-RFP23 strains were also 1.35 (http://broadinstitute.github.io/picard/). The Genome used in this study as readouts for nephrocyte morphology Analysis Toolkit version 2.6–4 was used for base quality score and function. recalibration, indel realignment, and variant discovery (single- nucleotide variants and indels). Read mapping and variant call- cDNA Cloning and Generation of NUP160 Transgenic ing were used for data analysis and interpretation. Variants were Fly Strains annotated with SeattleSeq SNP Annotation 150 (http://snp.gs. Awild-typehumanNUP160 cDNA encoding the common washington.edu/SeattleSeqAnnotation150/). Variants present 1437-amino acid NUP160 isoform (NCBI reference sequence at a frequency of .0.01 in the database of single-nucleotide NP_056046.1) was obtained from OriGene (catalog no. E803K R11733 polymorphisms (dbSNP; dbSNP 151: http://www.ncbi.nlm. RC218855). NUP160 and NUP160 cDNAs were nih.gov/SNP/) and the NHLBI GO Exome Sequencing Project generated from the wild-type allele using the QuikChange or present from local exomes of unaffected individuals were Site-Directed Mutagenesis Kit (Clontech) and sequenced to excluded. Variants were filtered with the in-house Variant confirm that no other mutations were induced except for the Analysis and Filtration Tool software. Candidate genetic var- intended variation at glutamic acid 803 and the truncation at iants in the NUP160 gene were confirmed by Sanger sequenc- arginine 1173, respectively. The cDNAs were individually ing on an ABI 3730XL DNA Analyzer (Shanghai Invitrogen cloned into the pUAST-attB vector, and the transgenes were Biotechnology Co.). introduced into a fixed chromosomal docking site by germ- line transformation to ensure that the wild-type NUP160 allele Variant Filtering and the two variant alleles were expressed at the exact same The following criteria were used for variant filtering: (1) rare level in the nephrocytes. variants with ,0.01 allele frequency in control databases, including dbSNP 151 and Exome Variant Server; (2)homo- RFP Uptake Assay zygous or compound-heterozygous variants; (3)nonsynonymous Flies from the MHC-ANF-RFP, Hand-GFP, and Dot-Gal4 variants affecting the coding sequence, obligatory splice accep- transgenic lines were crossed with flies from the UAS- tor and donor site mutations, and in-frame coding deletion/ Nup160-RNAi transgenic lines at 25°C.23 One day after egg insertions; (4) pathogenic or likely pathogenic revealed by web- laying, embryos were shifted to 29°C. RFP uptake by pericar- based mutation analysis prediction tools, including PolyPhen-2, dial nephrocytes was assessed in second instar larvae or adult SIFT, PROVEAN, and MutationTaster; (5)variantsoutside flies 1 day postemergence by dissecting heart tissue into the above criteria discarded; and (6) mutations of interest con- Schneider Drosophila Medium (ThermoFisher) and examin- firmed by Sanger sequencing in the proband, the healthy sibling, ing cells using fluorescence microscopy. For quantifica- and the parents. tion, $20 nephrocytes were analyzed from each of three For additional annotative information about potential female flies per genotype. The results are presented as genes of interest, we also examined genomic, proteomic, mean6SD. A paired t test was used to analyze the data. Statistical genetic,andfunctionalinformationoncandidategenesusing significance was defined as P,0.05. databases, including GeneCards (http://www.genecards. org), Uniprot (http://www.uniprot.org), Ensembl (http:// Dextran Uptake Assay ensembl.org), and the Human Protein Atlas (http://www. Flies of the appropriate genotypes were allowed to lay eggs proteinatlas.org), as well as literature searches to help decide at 25°C. One day after egg laying, embryos were shifted to potential pathogenicity. Mouse Genome Informatics (http:// 29°C. Dextran uptake by pericardial nephrocytes was as- www.informatics.jax.org) was used for establishing whether sessed ex vivo in third instar larvae or adult flies 1 day post- SRNS disease phenotypes might be found in related mouse emergence by dissecting heart tissue into Schneider Drosophila models. Medium and examining the cells by fluorescence microscopy after 20 minutes incubation with AlexaFluo568-Dextran Fly Strains (10 kD; 0.05 mg/ml). For quantification, $20 nephrocytes Flies were reared on standard food at room temperature or 29° were analyzed from each of three female flies per genotype. C for UAS-GAL4 strains. The Dot-Gal4 strain23 was obtained The results are presented as mean6SD. A paired t test was from the Bloomington Drosophila Stock Center (BDSC). used to analyze the data. Statistical significance was defined Transgenic RNA interference (RNAi) lines were obtained as P,0.05.

nucleotides and amino acids are highlighted (red). Arrows indicate positions of mutations in relation to exons and protein motifs (in A and B, respectively). Relevant sequences from surviving family members (I1, father [F]; I2, mother [M]; II5, sister [S]) are also shown. (E) Phylogenetic comparisons of amino acid sequences of Nup160 protein regions affected by the identiﬁed mutations.

4 Journal of the American Society of Nephrology J Am Soc Nephrol 30: ccc–ccc,2019 www.jasn.org BASIC RESEARCH

Silver Nitrate Uptake Assay A renal biopsy was performed for the second time in 2002. Flies of the appropriate genotype were allowed to lay eggs on Light microscopy revealed nine glomeruli with segmental scle- standard apple juice plates for 24 hours. Freshly emerged first rosis (in one glomerulus), mild segmental mesangial pro- instar larvae were transferred to agar-only plates supplemented liferation (in eight glomeruli), renal tubular atrophy, focal with regular yeast paste containing silver nitrate (AgNO3;2.0g fibrous proliferation, and inflammatory cell infiltrates in the yeast in 3.5 ml 0.0005% AgNO3 solution) and allowed to de- tubulointerstitium but no lesions in small vessels (Figure 2C). 23 velop at 29°C until adulthood. AgNO3 uptake by pericardial IF showed scattered mesangial deposits of IgA (+++) but nephrocytes was assessed in third instar larvae or adult flies without deposit of IgM, IgG, C3, C4, C1q, Fibrin, k,andl 1 day postemergence by dissecting heart tissues into Schneider in five glomeruli. Electron microcopy showed effacement of Drosophila Medium and examining nephrocytes by phase podocyte foot processes, partial thickened and sclerosing glo- contrast microscopy. For quantification, .20 nephrocytes merular basement membrane, mesangial hypercellularity, were analyzed from each of three female flies per genotype. and subendothelial deposition of an electron dense material The results are presented as mean6SD. A paired t test was (Figure 2D). used to analyze the data. Statistical significance was defined as P,0.05. Treatment Outcomes Treatments with prednisone, cyclophosphamide, and tripterygium Immunofluorescence Imaging and Confocal wilfordii glycosides all failed. The proband (II6) progressed to Microscopy ESRD at age 15 years old, received a kidney transplant at age Larvae and adult flies were dissected and fixed for 10 minutes in 16.4 years old, and is alive at this time. The parents (I1 and I2) 4% paraformaldehyde in PBS. Mab 414 (ab24609; Abcam) and and surviving sister (II5) are healthy. One sister (II1) and one anti-Lamin (ADL84.12; Developmental Studies Hybridoma brother (II4) died of unknown causes in early childhood. Bank) primary antibodies were used at 1:500 dilution followed The other two older siblings (II2 and II3) were affected with by Cy5-conjugated secondary antibodies (Jackson Lab). Con- SRNS, and both died of ESRD at age 17 years old (Figure 1C, focal imaging was performed with a Zeiss ApoTome.2 micro- Supplemental Table 1). scope using a 203 Plan-Apochromat 0.8 N.A. air objective. To quantitatively compare fluorescence intensities, common set- NUP160 Variants tings were chosen to avoid oversaturation. ImageJ Software Whole-exome sequencing of family members revealed 56,387 version 1.49 was used for image processing. single-nucleotide variants and 6383 small insertions and deletions with mapping statistics (Supplemental Table 3). Ac- cording to the criteria for variant filtering, most remaining RESULTS variants were excluded. Mutations in known genes associated with SRNS were also excluded (Supplemental Table 2). Six Patient Clinical Features variant genes remained (TTC12, DCANP1, KIAA1549, A 10-year-old girl (II6) was admitted to the Department of NUP160, DNAH5,andHYDIN) (Supplemental Table 4). Nephrology, Shanghai Ruijin Hospital for persistent pro- The index family pedigree (Figure 1C) displayed character- teinuria that began at age 7 years old. The proband (II6) istics typical of an autosomal recessive disease. According to presented normal BP and 3+ urinary albumin; 24-hour uri- the clinical phenotype, all of the patients (II2, II3, and II6) nary protein excretion was 8.34 g, and serum albumin was could be diagnosed as having nonsyndromic kidney disease. 24 g/L. Serum cholesterol was 4.55 mmol/L, serum creatine On the basis of kidney phenotype, number of homozygotes, was 53 mmol/L, and serum urea nitrogen was 4.2 mmol/L. and expression of genes in human glomerular cells, we ex- Audiometry was normal, and a skin test showed continuous cluded five of the remaining genes (TTC12, DCANP1, collagen IV. KIAA1549, DNAH5,andHYDIN)(SupplementalTable4). Compound-heterozygous variants E803K and R11733 of Histopathology NUP160 (RefSeq accession number NM_015231.2) were in A renal biopsy was performed for the first time in 1999 when trans configuration (Figure 1). Variant E803K was detected the proband (II6) was age 10 years old. Light microcopy re- in the father (I1) and the sister (II5), and variant R11733 vealed 14 glomeruli with global sclerosis (in one glomerulus), was detected in the mother (I2). The healthy surviving indi- segmental sclerosis (in two glomeruli), vessels with spheroidal viduals (I1, I2, and II5) are heterozygotes carrying a single hyaline degeneration (in two glomeruli), mesangial enlarge- mutated allele of either E803K or R11733, but the proband ment (in nine glomeruli), focal tubular atrophy, segmental (II6) carries two mutant alleles of NUP160 (Figure 1D). Two fibrous proliferation, and inflammatory cell infiltrates in the siblings (II2 and II3) with SRNS died earlier, and DNA sam- tubulointerstitium but no lesions of small vessels. Immuno- ples were not available for analysis (Figure 1C, Supplemental fluorescence (IF) showed scattered mesangial deposits of both Table 1). NUP160 was the only candidate gene in which the IgA (++) and IgM (+) but without deposits of IgG, C3, C4, C1q, variants were precisely cotransmitted with the underlying Fibrin, k,andl in five glomeruli. recessive disease status and in addition, were not detected in

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PASM TEM AB

BM Control

50 µm 2 µm

C D Patient

50 µm 2 µm

Figure 2. The proband’s renal tissue histology and cell ultrastructure revealed FSGS. (A and B) Control normal glomerulus tissue samples were obtained from a patient with a kidney tumor. (C and D) Patient tissues were obtained from the proband (patient II6) (Figure 1C). (A and C) Light microscopic examination of periodic acid–silver metheramine (PASM)–stained glomerulus showing focal segmental glomerular hyaline degeneration and sclerosis in the patient sample. (B and D) Transmission electron microscopy (TEM) revealed effacement of podocyte foot processes and partial thick and sclerosing glomerular basement membrane (BM; arrows) in the patient sample. Magniﬁcation, 3400 in A and C; 312,000 in B and D.

520 control subjects. R11733 results in premature termina- that it was required for maintenance of normal adult tion of translation and production of a truncated protein. lifespan. E803K is predicted to be disease-causing by MutationTaster Tostudy nephrocyte physiologic functional deficits induced and neutral by Polyphen, SIFT, and PROVEAN (Supplemental by Nup160 gene silencing, we examined in vivo uptake of RFP Table 4), and it was also identified in another unrelated Chi- from the hemolymph in nephrocytes of larvae (Figure 3C) and nese family.11 To provide functional evidence to validate the adult flies (Figure 3E). In larval nephrocytes, Nup160 gene NUP160 mutations as pathogenic, we used a Drosophila car- silencing was associated with approximately 2.5-fold less diac nephrocyte experimental model to study the effects of RFPuptakethanobservedincontrollarvae.Inadultflies, NUP160 gene deficiency in vivo. fluorescence microscopy confirmed complete absence of nephrocytes and consequently, no RFP uptake (Figure 3E). Drosophila Nup160 Gene Silencing Functional phenotypic analysis was extended to a nephrocyte NUP160 is highly conserved from flies to humans (Figure 1E), sequestration assay in which larvae are raised on a diet sup- and it is constitutively expressed in all cell types. To study plemented with AgNO3.Innormalflies, ingested toxic AgNO3 NUP160 renal phenotypes in Drosophila,wefirst used accumulated and was sequestered in cardiac nephrocytes RNAi-based gene silencing to lower expression of the en- (Figure 3D). When nephrocyte Nup160 gene expression dogenous fly Nup160 gene specifically in nephrocytes. was silenced, sequestered AgNO3 was reduced by approxi- Nup160 gene silencing significantly reduced the lifespan mately fivefold in larvae (Figure 3D). Drosophila Nup160 of adult flies (Figure 3A). In larval stage, animals’ total num- gene silencing in nephrocytes, therefore, led to significant bers of nephrocytes were significantly reduced compared functional deficits in vivo and was associated with reduced with controls. Adult flies were found to lack nephrocytes adult flysurvival. entirely (Figure 3B). These observations indicated that To better understand the effects of Nup160 silencing on Nup160 was essential for nephrocyte survival during imma- renal cells, we examined larval nephrocytes by fluores- ture stages of development through adult emergence and cence microscopy using structural and functional markers

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A B * *

100 60 *** 50 80 40 60 30 40 Control 20 Survival(%) Nup160-IR

20 Nephrocyte Number 10

0 0 0 1020304050 55h larvae 3rd instar 1-day-old Time (Days) larvae adult Control Nup160-IR

C 1.2 * 1.0 0.8 Control 0.6

Uptake 0.4

55h Larva 0.2 Larval Nephrocyte RFP 0 Nup160 -IR Control Nup160-IR

3 1.2 *

3 1.0 0.8 Control 0.6

Uptake 0.4 Uptake 0.2

Larval Nephrocyte AgNO 0 Nup160 -IR

Larval Nephrocyte AgNO Control Nup160-IR

E Control 1-day-old Adult Nup160 -IR

Figure 3. Nephrocyte-specific silencing of endogenous Nup160 expression led to shortened adult fly lifespan, fewer nephrocytes, and reduced nephrocyte function. Nup160-IR flies carry a UAS-Nup160-RNAi transgene plus a nephrocyte-specific Dot-GAL4 driver that together silence endogenous fly Nup160 expression. Control flies carry only the Dot-GAL4 driver. (A) Survival curves for Nup160-IR and control adult flies (newly emerged on day 0). Nephrocyte-specific silencing of endogenous Nup160 expression was associated with reduced lifespan: Nup160-IR flies experienced 50% mortality at day 25 versus day 40 for control flies. (B) Nephrocyte-specific silencing of Nup160 induced progressive loss of nephrocytes during larval- to adult-stage development. Nephrocytes were counted in second instar (55-hour larvae) and third instar larval stages and adult flies within 24 hours postemergence. Progressively fewer nephrocytes were observed during larval-stage development, and nephrocytes were entirely absent in adult flies. For quantification, nephrocytes

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(Figure 4). Freshly dissected nephrocytes were functionally Overexpression of Wild-Type or Mutant Human assayed ex vivo for uptake of FITC-labeled 10-kD Dextran NUP160 Transgenes Did Not Induce Nephrocyte beads. Control nephrocytes readily took up the beads, re- Abnormalities sulting in abundant distinct pericytoplasmic vesicular fluo- Nephrocyte phenotypes induced by human NUP160 transgene rescence. NUP160 silencing severely reduced Dextran expression were analyzed. Nephrocyte-specific overexpression WT uptake, and limited fluorescence was mostly concen- of human NUP160 cDNA did not affect RFP uptake, and trated in the nephrocyte cell interior in clumped aggre- nephrocyte morphology was unchanged compared with gates; 49,6-diamidino-2-phenylindole staining showed nephrocytes of control flies (Supplemental Figure 1). Over- E803K R11733 that Nup160 silencing induced reduction in nuclear expression of NUP160 or NUP160 cDNA trans- volume. The profound effect of Nup160 silencing on the genes in nephrocytes did not lead to significant changes in nephrocyte nucleus was clearly shown by Mab414 IF label- nephrocyte function. These results indicate that neither of ing of the NPC. In normal nephrocytes, Mab414 distinctly these two mutant alleles encodes a gain-of-function or dom- and concisely labeled the nuclear circumference. Nup160 inant negative NUP160 mutation. silencing, by contrast, led to Mab414 labeling NPC components dispersed within the shrunken nuclear com- Rescue of Endogenous NUP160 Deficiency by partment (defined by 49,6-diamidino-2-phenylindole co- “Replacement” Human Transgene Expression labeling) (Figure 4A), presumably the result of failed The Drosophila model allows for a highly efficient “gene re- NPC assembly. The defective nuclear structure induced placement” approach to assess the relative abilities of wild- by Nup160 silencing was further confirmed by anti-Lamin type and mutant versions of human genes to rescue the antibody immunolabeling in which the normal (control) nephrocyte phenotypes induced by silencing of endogenous circumferential nuclear lamin localization appeared in- fly gene homologs. In this way, specific patient-derived mu- stead very irregular (Figure 4B). Drosophila Lamin gene tant alleles identified through genome sequencing can be silencing induced similar nephrocyte morphologic changes functionally implicated (i.e., validated) in renal cell dysfunc- (i.e., reduced nucleus) and functional deficits (reduced tion.27 We first coexpressed a wild-type human NUP160 Dextran uptake). Lamin silencing, however, did not lead cDNA transgene specifically in nephrocytes while simulta- to aberrantly localized Mab414 IF (Figure 4A). Nephrocyte neously silencing endogenous fly Nup160 expression. As functional defects induced by Nup160 gene silencing were, shown in Figure 5B, wild-type human NUP160 expression therefore, associated with striking changes in nephrocyte completely rescued Nup160-IR–induced absence of adult cellular morphology, presumably the result of severe break- nephrocytes, and the rescued nephrocytes were fully func- down of normal nucleocytoplasmic trafficking due to dis- tional. By contrast, neither the patient-derived NUP160 mu- rupted NPC localization caused by failed complex assembly tant allele E803K nor R11733 was capable of rescuing the in the absence of sufficient Nup160 protein. adult nephrocyte phenotype (Figure 5C). were counted from each of five larvae or adult flies of each genotype. The results are presented as mean6SD. *Statistical significance was defined as P,0.05. (C) Nephrocyte-specific silencing of Nup160 led to reduced uptake of fluorescently labeled marker protein. All larvae carry an MHC-ANF-RFP transgene expressing ANF-RFP marker protein in muscle cells, which is secreted into the flyhemolymph;itis normally taken up by and accumulated in nephrocytes (red fluorescence) before protein breakdown and recycling of amino acids. In addition, all flies carry a Hand-GFP transgene expressing a nuclear-localized GFP marker (green; predominantly nuclear fluorescence) in both pericardial nephrocytes (larger nuclei with faint cytoplasmic fluorescence) and cardiomyocytes (smaller nuclei). Fluorescence microscopy revealed that, in control 55-hour larvae, almost every nephrocyte took up and accumulated abundant ANF-RFP marker protein from the larval hemolymph (left panels; center panels show the boxed areas). By contrast, nephrocytes of Nup160-IR larvae expressing the Nup160-RNAi transgene showed reduced levels of ANF-RFP overall, although a minority of nephrocytes displayed essentially normal levels of RFP. Quantification of ANF-RFP uptake by control versus Nup160-IR nephrocytes is shown in right panel. Nephrocyte RFP uptake was reduced approximately 2.5-fold as a result of Nup160 gene silencing; $20 nephrocytes were analyzed from each of five larvae per genotype. The results are presented as mean6SD. *Statistical significance was defined as P,0.05. (D) Ingested silver nitrate (AgNO3) taken up and sequestered by third instar larval nephrocytes revealed by phase contrast microscopy. Upper left and upper center panels show abundant AgNO3 within control nephrocytes, whereas lower left and lower center panels show very reduced levels in Nup160-IR nephrocytes delineated by the dotted outline in left panels (center panels show higher-magnification views of boxed nephrocytes).

Quantification of AgNO3 in control versus Nup160-IR nephrocytes is shown in right panel. Nephrocyte AgNO3 was reduced nearly fivefold as a result of Nup160 gene silencing; $20 nephrocytes were analyzed from each of five larvae per genotype. The results are presented as mean6SD. *Statistical significance was defined as P,0.05. (E) Nup160 gene silencing led to complete absence of adult fly nephrocytes. All flies express MHC-ANF-RFP and Hand-GFP transgenes. Fluorescence microscopy showed control adult nephrocytes with abundant cytoplasmic ANF-RFP (red) and nuclear GFP (green) as well as cardiomyocytes (smaller green nuclei; no RFP fluorescence). By contrast, no nephrocytes were observed in Nup160-IR flies expressing a UAS-Nup160-RNAi driven by Dot-Gal4, although cardiomyocytes (GFP-positive nuclei) were normal. Right panels show higher magnification views of boxed areas. *Normal locations of nephrocytes relative to cardiomyocytes.

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A Mab414 Dextran DAPI Merge Control Nup160 -IR Lam -IR

B Lamin Dextran DAPI Merge Control Nup160 -IR

Figure 4. Nup160 gene silencing induced nuclear pore complex (NPC) and lamin abnormalities in Drosophila nephrocytes. (A) Mab414 (green) immunolabeled components of the NPC, which in control nephrocytes, generated a sharp and highly regular ring around the nucleus (indicated by 49,6-diamidino-2-phenylindole [DAPI] staining; blue). In nephrocytes of larvae in which Nup160 gene expression was silenced (Nup160-IR), by contrast, Mab414 immunolabeling was dispersed across the nucleus, and it was more abundant in the cytoplasm, indicating abnormal localization of NPC components. Normal ex vivo uptake of FITC-labeled 10-kD Dextran particles (red) was significantly affected by Nup160 gene silencing, indicating functional nephrocyte defects linked to abnormal NPC localization. Silencing of the Drosophila Lamin gene by nephrocyte-specificexpressionofaUAS-Lam-RNAi transgene driven by Dot-Gal4 (Lam-IR) led to marked abnormalities in nuclear size and shape and a severe functional deficit, but NPC localization appeared normal. (B) Mab ADL67.10 labels all isoforms of Drosophila Sf9 Lamin but does not label Lamin C. In control nephrocytes, Lamin (green) appeared as a sharp regular ring around the nucleus. Nup160 gene silencing (Nup160-IR), by contrast, did not disrupt circumferential nuclear Lamin localization (although nuclear morphology was altered as indicated by DAPI staining), but labeling was less concise and uniform than in control nephrocytes.

R11733 In third instar larvae, wild-type human NUP160 expression whereas the NUP160 transgene did not (Figure 6). completely rescued the ﬂy gene silencing–induced defects in This result suggests that the E803K missense allele pro- nuclear volume, Dextran uptake, NPC localization, and duces an Nup160 protein that can promote NPC assembly nuclear lamin morphology (Figure 6). By contrast, neither and support nuclear lamin and potentially, nuclear mem- the patient-derived NUP160 mutant allele E803K nor brane morphology but that still results in defective nucleocy- R11733 was capable of rescuing defective nuclear volume toplasmic transport, with severe consequences for nephrocyte E803K or Dextran uptake. The NUP160 mutant transgene did cellular structure and function. The truncated R11733 allele, by rescue NPC localization and nuclear lamin morphology, contrast, is essentially null.

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A Control

B Nup160-IR Nup160-IR + NUP160-wt

C Nup160-IR +NUP160-E803K Nup160-IR +NUP160-R1173X

Figure 5. Adult nephrocyte phenotypes induced by Nup160-IR were rescued by wild type human NUP160 transgene, but not by NUP160E803K or NUP160R1173X. (A) Fluorescence micrograph shows nephrocytes of adult flies 1 day postemergence. ANF-RFP fluorescence (red) is merged with GFP (green; mostly nuclear). A GFP transgene is expressed under the control of a Hand gene enhancer (Hand-GFP)toconfirm pericardial nephrocyte cell identity. All flies are transgenic for Hand-GFP. Control flies carry the Dot-Gal4 driver but no RNA interference construct. (B, left panel) Nephrocyte-specific Nup160-IR transgene expression silenced endogenous fly Nup160, leading to complete absence of adult nephrocytes. Unaffected cardiomyocytes exhibited GFP fluorescence. (B, right panel) Nephrocyte-specific expression of a wild-type human NUP160 transgene (NUP160-wt) rescued the adult nephrocyte phenotype induced by Nup160-IR. (C, left panel) Nephrocyte-specific expression of a mutant E803K NUP160 transgene (NUP160-E803K) failed to rescue the adult nephrocyte phenotype induced by Nup160-IR. (C, right panel) Nephrocyte-specific expression of a mutant R11733 NUP160 transgene (NUP160-R11733) failed to rescue the adult nephrocyte phenotype induced by Nup160-IR.

DISCUSSION genes through bioinformatics-based analysis of minor allele frequency, mutation type, clinical characteristics, and web- The findings in this study indicate that compound-heterozygous based pathogenic prediction (Supplemental Table 4). We ulti- R11733 E803K mutations, NUP160 and NUP160 , in the proband mately focused on compound-heterozygous mutations R11733 E803K with familial SRNS are likely pathogenic, which implies that NUP160 and NUP160 on the basis of the proband NUP160 mutations are associated with SRNS. having only renal manifestations without syndromic disorder The pedigree of this study delineated an autosomal recessive associated with extrarenal manifestations. The residues af- family with SRNS (Figure 1C), which consisted of healthy fected in these NUP160 mutant alleles are largely conserved. R11733 parents (I1 and I2), the proband (II6), two siblings (II2 and NUP160 changes an arginine codon to a stop codon. E803K II3) affected with SRNS, two siblings (II1 and II4) dying of The NUP160 mutation is predicted to be disease causing unknown causes, and one healthy sibling (II5). The proband’s by MutationTaster. Finally, we functionally validated the renal specimen revealed FSGS (Figure 2), and IF staining NUP160 mutations in vivo as potentially implicated in SRNS showed nonspecific scattered mesangial deposits of IgA and pathogenicity. IgM. The proband (II6) progressed to ESRD and underwent We used the Drosophila nephrocyte as an experimental po- renal transplantation. Until now, a 13-year follow-up of the docyte model. The nephrocyte has previously been shown to graft kidney shows normal renal function and no post-transplant share striking molecular, cellular, structural, and functional reoccurrence of nephrotic syndrome. homologies with mammalian podocytes, and it has been Here, on the basis of genomic sequencing, we excluded the used previously to validate candidate renal disease genes as possibility that the proband carried mutations in genes already well as specific patient-derived mutant alleles.8,22,25,27,28 known to be associated with SRNS (Supplemental Table 2). We Here, we showed that Nup160 is essential for normal nephrocyte systematically reduced the candidate gene list to six variant cell maintenance/survival through adult-stage development and

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A Mab414 Dextran DAPI Merge Control Nup160 -IR NUP160-wt Nup160 -IR+ Nup160- IR+ NUP160-E803K Nup160- IR+ NUP160-R1173X

Figure 6. Third instar larval nephrocyte phenotypes induced by Nup160-IR were rescued by wild type human NUP160,butnotby NUP160E803K or NUP160R1173X. (A) Mab414 (green) immunolabeled components of the nuclear pore complex (NPC), ﬂuorescent Dextran uptake indicated nephrocyte function (Figure 4), and 49,6-diamidino-2-phenylindole (DAPI) staining revealed nuclear position and overall morphology. (B) Mab ADL67.10 labels all isoforms of Drosophila Sf9 Lamin (green) but does not label Lamin C. Nephrocyte- speciﬁc expression of a wild-type human NUP160 transgene (NUP160-wt) rescued all larval nephrocyte phenotypes. An NUP160E803K 3 mutant transgene (NUP160-E803K) rescued NPC localization and Lamin morphology but not Dextran uptake. A NUP160R1173 mutant transgene (NUP160-R11733) failed to rescue any larval nephrocyte phenotypes.

E803K R11733 that it is furthermore required for normal adult fly lifespan. NUP160 ,andNUP160 alleles using a “gene re- WT Nephrocyte function as assayed by uptake of protein, AgNO3, placement” approach. NUP160 was shown to completely and Dextran particles was severely impaired as a result of rescue the entire spectrum of nephrocyte phenotypes induced R11733 Nup160 gene silencing. Affected nephrocytes exhibited reduced by endogenous fly Nup160 gene silencing. NUP160 ,by nuclear volume, NPC components were dispersed, and nuclear contrast, exhibited no phenotype rescue, consistent with this E803K lamin localizationwas irregular. These observations indicate that being a null allele. The missense NUP160 mutant allele Nup160 is essential for NPC assembly and nuclear structure to did rescue NPC component localization and lamin morphol- support nucleocytoplasmic trafficking required for normal cell ogy defects but not defects in other phenotypes, suggesting morphology and function. that this mutation can promote NPC assembly and localiza- The nephrocyte model further allowed us to examine dif- tion but still severely impair nucleocytoplasmic transport and WT ferential phenotypic rescue potential of human NUP160 , other NPC-associated functions required for normal

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B Lamin Dextran DAPI Merge Control Nup160 -IR NUP160-wt Nup160 -IR+ Nup160- IR+ NUP160-E803K Nup160- IR+ NUP160-R1173X

Figure 6. Continued. nephrocyte physiology. Nephrocyte-speciﬁc overexpression of respectively.4–6 Mutations in ACTN4 change the cytoskeletal missense and truncated mutant human alleles did not in- dynamics of podocytes and lead to adult-onset autosomal duce functional defects, suggesting that these two mutant dominant FSGS.7 Moreover, in vertebrates, transmembrane allelesarenotgain-of-functionordominantnegativemuta- Nups anchor the NPCs within the nuclear envelope through tions. Together, the experimental observations indicate that interactions with two major scaffold modules: the symmetri- E803K R11733 NUP160 and NUP160 mutations are pathogenic cally localized Nup107–160 complex and the central Nup93 mutations in the patient with SRNS and FSGS described in complex. Nup107–160 complexes are composed of nine dis- this study. tinct subunits: Nup160/Nup120, Nup133, Nup107, Nup96, We recently found that knockdown of NUP160 inhibited Nup85/Nup75, Nup43, Nup37, Seh1, and Sec13. In contrast cell proliferation; induced apoptosis, autophagy, and cell mi- to dynamic peripheral Nups, the components of the Nup107– gration of mouse podocytes cultured in vitro; and altered the 160 complex are very stable and anchored within the NPCs expression and localization of podocyte-associated molecules, during interphase.12,13 Signiﬁcantly, previous reports have pro- including nephrin, podocin, CD2AP, and a-actinin-4.14 Mu- vided functional evidence from model system experiments that tations in NPHS1, NPHS2,andCD2AP disrupt the integrity of NPC-related mutations in NUP85,11 NUP93,9 NUP107,10,11 the slit diaphragm and lead to congenital nephrotic syndrome, NUP133,11 and NUP2059 genes are causal for SRNS. Together, early-onset autosomal recessive SRNS, and early-onset FSGS, these data suggest that two compound-heterozygous mutations,

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E803K R11733 NUP160 and NUP160 , caused familial SRNS in the Supplemental Figure 1. Overexpression of wild-type and mutant patient analyzed in this study. human NUP160 transgenes. Two limitations of this study should be noted. First, renal Supplemental Table 1. Clinical features of three patients in a biopsy tissues taken from the patient (II6) 16 years ago were not nonconsanguineous Chinese family with steroid-resistant nephrotic analyzed for expression of NUP160. Second, only one patient syndrome. with SRNS was identified with compound-heterozygous mu- Supplemental Table 2. A list of known genes associated with steroid- tations in NUP160. Recently, however, Braun et al.11 reported resistant nephrotic syndrome. compound-heterozygous potentially pathogenic mutations Supplemental Table 3. Mapping statistics of whole-exome E803K R9103 NUP160 and NUP160 in two siblings with SRNS sequencing. and proteinuria from another nonconsanguineous Chinese Supplemental Table 4. Rare variants found in patient II6. family. The older brother presented at age 16 years old with nephrotic syndrome that was resistant to therapy with steroids or other immunosuppressants. His biopsy revealed FSGS, and REFERENCES his renal function corresponded to stage 3 CKD. The sister presented at age 7 years old with proteinuria. Neither sibling 1. Wiggins RC: The spectrum of podocytopathies: A unifying view of exhibited extrarenal symptoms. Interestingly, the missense glomerular diseases. Kidney Int 71: 1205–1214, 2007 E803K mutation NUP160 found in the Chinese family in the 2. Trautmann A, Bodria M, Ozaltin F, Gheisari A, Melk A, Azocar M, et al.; PodoNet Consortium: Spectrum of steroid-resistant and congenital et al 11 work by Braun . isthesameasthatinthefamilyin nephrotic syndrome in children: The PodoNet registry cohort. Clin J Am our study, whereas these two Chinese families with SRNS Soc Nephrol 10: 592–600, 2015 are not closely related. 3. 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