Novel UMOD mutations in familial juvenile hyperuricemic nephropathy lead to abnormal uromodulin intracellular trafficking

Gene 531 (2013) 363–369

Contents lists available at ScienceDirect

Gene journal homepage: www.elsevier.com/locate/gene

Novel UMOD mutations in familial juvenile hyperuricemic nephropathy lead to abnormal uromodulin intracellular trafficking Maojing Liu a, Yuqing Chen a,⁎, Yu Liang a, Ying Liu a, Suxia Wang a,b, Ping Hou a, Hong Zhang a, Minghui Zhao a a Renal Division, Department of Medicine, Peking University First Hospital, Institute of Nephrology, Peking University, Key Laboratory of Renal Disease, Ministry of Health of China, Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Ministry of Education, Beijing 100034, China b Laboratory of Electron Microscopy, Peking University First Hospital, Beijing 100034, China

a r t i c l e

i n f o

Article history: Accepted 12 August 2013 Available online 27 August 2013 Keywords: UMOD Familial juvenile hyperuricemic nephropathy Uromodulin Tamm–Horsfall protein FJHN

a b s t r a c t Background: Familial juvenile hyperuricemic nephropathy (FJHN) is an autosomal dominant disorder characterized by hyperuricemia and progressive chronic kidney disease. Uromodulin gene (UMOD) mutations, leading to abnormalities of uromodulin intracellular trafficking contribute to the progress of the disease. Methods: We did UMOD screening in three Chinese FJHN families. We thus constructed mutant uromodulin express plasmids by site-mutagenesis from wild type uromodulin vector and transfected them into HEK293 (human embryonic kidney) cells. And then we detected uromodulin expression by western blot and observed intracellular distribution by immunofluorescence. Results: We found three heterozygous mutations. Mutation Val109Glu (c.326T/A; p.Val109Glu) and mutation Pro236Gln (c.707C/A; p.Pro236Gln) were newly indentified mutations in two distinct families (family F1 and family F3). Another previously reported UMOD mutation Cys248Trp (c.744C/G; p.Cys248Trp) was detected in family F2. Phenotypes varied both within the same family and between different families. Uromodulin expression is abnormal in the patient biopsy. Functional analysis of mutation showed that mutant types of uromodulin were secreted into the supernatant medium much less when compared with wild type. In mutant type uromodulin transfected cells, intracellular uromodulin localized less in the Golgi apparatus and more in endoplasmic reticulum(ER). Conclusions: Our results suggested that the novel uromodulin mutations found in the Chinese families lead to misfolded protein, which was retained in the endoplasmic reticulum, finally contributed to the phenotype of FJHN. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Familial juvenile hyperuricemic nephropathy (FJHN; MIM 162000) and medullary cystic kidney disease type 2 (MCKD2; MIM 603860) are autosomal dominant tubulointerstitial nephropathy, characterized by hyperuricemia, hypertension and progressive renal disease. Allelic disorders, FJHN and MCKD2 are collectively referred to uromodulin-associated kidney disease (UAKD) (Duncan and Dixon, 1960; Hart et al., 2002; Scolari et al., 1999). Uromodulin, also known as Tamm–Horsfall glycoprotein, is the most abundant protein in human urine (Kumar and Muchmore, 1990). Uromodulin, a 640-amino-acid protein, is heavily glycosylated

Abbreviations: UMOD, uromodulin gene; FJHN, Familial juvenile hyperuricemic nephropathy; MCKD2, medullary cystic kidney disease type 2; UAKD, uromodulin-associated kidney disease; ER, endoplasmic reticulum; EGF-like, epidermal growth factor like; D8C, eight cysteine domain; ZP, zona pellucid; GPI, glycosylphosphatidylinositol; TAL, thick ascending limb; DCT, distal convoluted tubule; HEK293, human embryonic kidney 293; CRF, chronic renal failure; SDS-PAGE, Sodium dodecylsulfate-polyacrylamide gel electrophoresis; FEUA, Fraction of excretion of uric acid; ESRD, end stage renal disease; GFR, glomerular filtration rate; NF-κB, nuclear factor-κB; JNK, c-Jun N-terminal kinase. ⁎ Corresponding author. Tel.: +86 10 83572388; fax: +86 10 66551055. E-mail address: [email protected] (Y. Chen). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.08.041

(30% of molecular weight) (van Rooijen et al., 1999). It contains an N-terminal signal peptide, epidermal growth factor like (EGF-like) domains, an eight cysteine domain (D8C), a zona pellucid (ZP) domain and a glycosylphosphatidylinositol (GPI) anchor segment. Uromodulin is exclusively synthesized by the cells of the thick ascending limb (TAL) and early distal convoluted tubule (DCT) of the kidney (SerafiniCessi et al., 2003). It is produced in the endoplasmic reticulum (ER), shuttled to the Golgi apparatus for further decoration, then to the apical cell membrane as a GPI-linked molecule and finally released into the urine by proteolytic cleavage (Fukuoka and Kobayashi, 2001; Santambrogio et al., 2008; Serafini-Cessi et al., 1993). UMOD variations in coding region could lead to defects of protein folding, so that misfolded immature uromodulin was trapped in the ER, and uromodulin was finally less expressed or released by the apical cell membrane (Bernascone et al., 2006; Jennings et al., 2007; Smith et al., 2011; Vyletal et al., 2006; Williams et al., 2009). Nearly 60 variations have been reported to contribute to the disease so far. They were mainly localized in exon 3 and exon 4, which encoding the three EGF-like domains, as well as the D8C. Most of the reported variations were missense mutations or small in-frame deletions (Bollee et al., 2011; Lee et al., 2010; Nakayama et al., 2012; Smith et al., 2011; Vyletal et al., 2010; Wei et al., 2012).

364

M. Liu et al. / Gene 531 (2013) 363–369

Here we identified three UMOD variations, two novel mutations found by us and one reported by others, in Chinese FJHN families. We then transfected plasmids of wild-type and mutant uromodulin into HEK293 (human embryonic kidney) cells and explored whether the three UMOD variations had impact on uromodulin intracellular trafficking and secretion.

into 35 mm plates at a density of 2.5 × 105 cells. Cells were transiently transfected with 2 μg uromodulin expression plasmid using X-tremegene HP DNA Transfection Reagent (Roche, Germany). Transfective efficiency of plasmids was evaluated by GFP fluorescence of cells transfected. Expression of the UMOD–GFP fusion proteins was examined 48 h later by western blot and by confocal laser microscopy. Each experiment was repeated at least three times.

2. Materials and methods 2.1. Patients

2.5. Western blot

We ascertained three FJHN/MCKD families (Fig. 1 and Table 1) according to clinical criteria (Dahan et al., 2003): 1) a history of gout or hyperuricemia (N 6 mg/dl in female, 7 mg/dl in male); 2) a history of chronic renal failure (CRF) with/without inheritance compatible with an autosomal dominant trait; 3) diagnosis of MCKD by renal biopsy; and 4) exclusion of other common hereditary nephropathies. All participants provided informed consents.

Cell lysate and supernatant of cell culture were collected separately at 48 h after transfection. Proteins were quantified by the Micro BCA Protein Assay (Pierce, Rockford, IL, USA). Denatured proteins were separated by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Equal amounts of proteins were transferred electrophoretically to nitrocellulose membranes and membranes were rinsed and blocked with 5% skimmed milk. Membranes were first incubated with rabbit anti human Tamm–Horsfall protein (H-135, 1:500; Santa Cruz, CA, USA) and mouse anti human β-actin (1:500; Santa Cruz, CA, USA), and followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody, as well as HRP-conjugated goat anti-mouse secondary antibody (1:5000; Santa Cruz, CA, USA). The membranes were washed again and uromodulin expression was detected with Western Lightning Plus ECL (Perkin Elmer, MA, USA). Protein bands were quantified using Image J densitometry software (1.46r; National Institutes of Health, USA).

2.2. Mutation screening Genomic DNA was isolated from whole blood using a modified extraction technique (Miller et al., 1988). The genomic DNA reference sequence of UMOD (NC_000016.9, OMIM: 162000, Gene ID: 7369) was obtained from the Entrez gene database (http://www.ncbi.nlm. nih.gov/gene/7369). Primers for polymerase chain reaction amplification and sequencing were designed using Primer3 (http://frodo.wi. mit.edu/primer3/). The presence of mutations was confirmed by bidirectional sequencing of coding region and adjacent intronic segments of the UMOD. 2.3. Preparation of uromodulin expression vectors A pCMV-AC-GFP plasmid containing wild-type uromodulin cDNA was purchased from Origene Technologies Inc. (Rockville, MD, USA). Synthetic replacements (at mutation site) were made by QuikChange Site-Directed Mutagenesis Kit (Stratagene, CA, USA) to generate plasmids of mutant types of uromodulin. All plasmids were sequenceverified before use. 2.4. Cell culture and transfection HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) containing 5 mM glucose and 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) at 37 °C in a humidified atmosphere containing 5% CO2. HEK293 cells were seeded

Fig. 1. Schematic representation of uromodulin protein structure. Positions of uromodulin variants identified in this study were presented. The N-terminal region contains an N-terminal signal peptide, three epidermal growth factor (EGF)-like modules and a conserved cysteine residue (D8C). The C-terminal region includes the zona pellucida (ZP) and a phosphatidylinositol (GPI) anchor.

2.6. Immunofluorescence 8 × 104 of HEK293 cells were grown on 25 × 75 mm glass chamber slides (Nunc Lab-Tek II Chamber Slide System, Thermo Scientific, MA, USA) for 36 h and transfected as described above. At 48 h after the transfection, the cells were fixed with 100% methanol at −20°C for 10 min, permeabilized in 0.1% Triton X-100 for 5 min and blocked in 1% BSA for 1 h. Cells were then incubated overnight at 4 °C with rabbit polyclonal antibody against Calnexin—ER membrane marker (1:200, Abcam, Cambridge, UK) for ER localization or rabbit polyclonal antibody against Giantin—Golgi Marker (1:500, Abcam, Cambridge, UK) for Golgi localization. And then antibodies—Cy3-conjugated AffiniPure Donkey Anti-Rabbit IgG (H + L) (Jackson ImmunoResearch Laboratories, Inc., PA, USA) were used. Prepared slides were examined under a laser scanning confocal microscope (Olympus viewer 1000, Olympus, Japan).

2.7. Immunohistochemistry Immunohistochemical staining was performed on 4-μm deparaffinized sections of formaldehyde-fixed renal tissue using mouse anti-human uromodulin antibodies (1:400, Cedarlane, Canada). Sections were deparaffinized in xylene–ethanol at room temperature and rehydrated immediately in PBS, then immersed in citric acid buffer (0.01 M, pH 6.0) and were treated in an 800 W microwave oven for 2 min and then at 200 W for 9 min, for antigen retrieval. After washing in PBS for 10 min, sections were immersed into freshly prepared 3% hydrogen peroxide in methanol solution for 30 min at room temperature to quench endogenous peroxidase activity. To block nonspecific staining, sections were incubated with 3% BSA in PBS at room temperature for 30 min. The primary antibodies were added on each section and incubated overnight at 4 °C. And then the slides were exposed to secondary antibodies. Dako EnVision HRP (Dako A/S, Copenhagen, Denmark) was added and followed by 30 min incubation at 37 °C. Sections were developed in fresh hydrogen peroxide plus 3,3-diaminobenzidine tetrahydrochloride solution for 3 min. Finally, sections were counterstained with Mayer's hematoxylin.

M. Liu et al. / Gene 531 (2013) 363–369

365

Table 1 Synopsis of clinical data of the patients. Family

Gender/age

Age of onset

Pcr (mg/dl)

PUA (mg/dl)

Hypertension

FEUA (%)

Kidney biopsy

Renal ultrasound

Present status

Genotype

F1 I-1 II-1 II-2

F/47 y M/25 y M/18 y

NA NA 18 y

0.43 0.66 2.2↑

3.7 5.5 8.2↑

Yes No No

NA NA 5%↓

NA NA Chronic interstitial nephritis

NA NA Small cysts, calculus in left kidney

Asymptomatic Asymptomatic ESRD

Val109Glu Val109Glu Val109Glu

F2 II-1 II-2 III-1

M/+ F/49 y F/21 y

12 y 18 y 11 y

NA NA 0.93

NA NA 10.1↑

NA NA No

NA NA 5.6%↓

NA NA NA

NA NA Reduced parenchyma, small cysts

RRT/33 y, Death/35 y RRT/41 y Hyperuricemia

Cys248Trp Cys248Trp Cys248Trp

F3 III-4 III-6 III-9 IV-8 IV-9 IV-1 IV-3 IV-4 IV-6 IV-7 IV-10

F/51 y M/49 y F/39 y F/24 y M/20 y F/24 y F/21 y F/26 y M/20 y F/27 y M/15 y

NA NA 35 y 24 y NA NA NA NA NA NA NA

2.8↑ 1.5 ↑ 1.5 ↑ 2.8↑ 1.1 0.81 0.76 0.68 0.89 0.62 0.84

5.5 5.8 8.2↑ 10.1↑ 6.5 3.5 4.7 4.9 5.8 3.8 3.8

No No No Yes No No No No No No No

NA NA NA 6.2%↓ NA NA NA NA NA NA NA

NA NA NA NA NA NA NA NA NA NA NA

Normal Normal Normal Small kidneys Normal Normal Normal Normal Normal Normal Normal

CRF CRF Hyperuricemia, ESRD Hyperuricemia, ESRD Asymptomatic Normal Normal Normal Normal Normal Normal

WT WT Pro236Gln Pro236Gln Pro236Gln WT WT WT WT WT WT

Arrows denote increase vs decrease in relation to age-dependent normal values. NA, not available; y, years; Pcr, plasma creatinine; PUA, plasma uric acid; FEUA, fractional excretion of uric acid; CRF, chronic renal failure; ESRD, end stage renal disease; RRT, renal replacement therapy; +, Died; WT, wild-type.

2.8. Statistical analyses Statistical analyses were performed with SPSS 13.0 (SPSS Inc., Chicago, IL, USA). For parametrical data, a two-tailed one-way ANOVA was used, with Least Significant Difference (LSD) in case of equal variances and Tamhane's T2 in case of unequal variances. Crosstab test was used to compare the frequency of colocalization of uromodulin and ER/Golgi protein markers. The results were considered statistically significant at P b 0.05. 3. Results 3.1. Clinical and biochemical findings Nine individuals from the three families carried UMOD heterozygous mutations. Among them, six persons were identified to have hyperuricemia between 11 and 35 years of age and developed end stage kidney disease between 33 and 41 years old. Ultrasound examination of kidneys revealed small cysts around cortical-medullary junction in the two of them. Only one of them accepted renal biopsy and was diagnosed as medullary cystic kidney disease (Table 1, Fig. 2). F1-II-2 is an 18 year-old male without family history of renal disease. At the age of 18, he was found to have increased blood pressure (150/90 mm Hg), abnormal plasma creatinine level (195 μmol/l), hyperuricemia (489 μmol/l) and reduced urinary excretion of uric acid (FEUA 5%). The renal biopsy revealed medullary cystic kidney disease, although no cysts were identified by renal ultrasound. His renal function gradually decreased and progressed to end stage kidney disease three years after renal biopsy, His mother and elder brother have no history of hyperuricemia or increased plasma creatinine, although they were also identified as carriers of the same UMOD mutation. His mother showed hypertension, but she refused further examination. F2-III-1 is a 21 year old girl with family history of hyperuricemia and end stage kidney disease (ESKD). Her mother showed hyperuricemia at the age of 18 and received renal transplantation at 41 years old due to ESKD. Her uncle also had hyperuricemia, progressive kidney disease and died after renal transplantation. Her grandfather also died of uremia. The girl presented hyperuricemia at 11 years old, with reduced fraction excretion of uric acid. Her plasma creatinine has been kept

within normal range for more than 10 years. Ultrasound examination showed normal size kidneys with reduced parenchyma and small cysts. F3-IV8 is a 24-year-old female, presented hyperuricemia, hypertension and progressive renal failure. She has a big family. Three persons of the first and second generation died of uremia. Her mother died of breast cancer at 31 years old, and information about renal disease was not available. Her brother (IV9) has the same mutation but remain asymptomatic. There are 3 persons in the 3rd generation that have increased plasma creatinine. One of her aunt carries the same mutation and presented increased plasma creatinine as well as hyperuricemia. Another aunt (F3-III4) and an uncle (F3-III6) of F3-IV8 presented normal plasma uric acid level and mildly increased plasma creatinine, but mutations were not found in their UMOD gene.

3.2. Identification of UMOD mutations We identified two novel (c.326T/A; p.Val109Glu; c.707C/A; p.Pro236Gln) and one previously reported (c.744C/G; p.Cys248Trp) UMOD mutations in 9 of 17 members of the 3 families. All three mutations located in exon 3 of UMOD (Table 2, Fig. 1). In family F1, we identified a novel heterozygous missense mutation (c.326T/A; p.Val109Glu) altering the epidermal growth factor-like domain (cbEGF3) of UMOD. The patient's mother and elder brother also have the same heterozygous mutation without any apparent clinical symptoms. In family F3 (III-9, IV-8, IV-9), another novel heterozygous missense mutation 707C/A was detected, causing the amino acid exchange of Pro236Gln. This mutation affected D8C in the encoding region of UMOD. The mutation was not found in other family members (IV-1, IV-3, IV-4, IV-6, IV-7, IV-10) among those with available DNA. We also did not identify this mutation in the two members of the family (III-4, III-6) with mildly elevated plasma creatinine. In family F2, we found a previously reported UMOD mutation (c.744C/G; p.Cys248Trp), also affecting D8C of UMOD. The same mutation was also detected in the patient's mother and uncle. None of the three mutations were detected in 100 healthy controls.

366

M. Liu et al. / Gene 531 (2013) 363–369

Fig. 2. Variation screening of UMOD in the families. Genealogical trees of the families are shown. Black symbols denote clinically affected individuals. Gray symbols denote no-syndrome carriers of UMOD variation. Open symbols denote unaffected individuals. The upper sequence was the wild-type (WT) sequence and the lower sequence revealed the mutation (MUT) of UMOD in each family. a. Family F1. A heterozygous missense mutation of 326T/A (p. Val109Glu) was identified. b. Family F2. A heterozygous missense mutation of 744C/G (p.Cys248Trp) was detected. c. Family F3. A heterozygous missense mutation of 707C/A (p.Pro236Gln) was detected.

3.3. Delayed maturation and trafficking defects of mutant uromodulin To explore whether the three identified mutations of UMDO gene influenced the intracellular trafficking of uromodulin, we got mutant uromodulin express vectors of Val109Glu, Pro236Gln, and Cys248Trp by site mutagenesis from wild type uromodulin-GFP plasmid, and transfected them into HEK293 cells. After 48 h of cell culture, supernatant and cell lysate were collected. Transfective efficiency was about 50%. Both immature uromodulin (ER resident) and mature uromodulin were detected in cells transfected with wild type and mutant type uromodulin (Fig. 3a, c). The ratio of mature over immature uromodulin (M–P ratio) was obviously higher in cells transfected with wild type uromodulin vectors than in cells transfected with mutant types (Fig. 3b). Consequently mutant uromodulins secreted into the supernatant medium were much less in mutant type compared with wild type Table 2 UMOD mutations in FJHN families. Family

Nucleotide a

Exon

Amino acid change

Uromodulin domain affected

F1 I-1 II-1 II-2

T326A T326A T326A

3 3 3

Val109Glu Val109Glu Val109Glu

EGF3 EGF3 EGF3

F2 II-1 II-2 III-1

C744G C744G C744G

3 3 3

Cys248Trp Cys248Trp Cys248Trp

D8C D8C D8C

F3 III-9 IV-8 IV-9

C707A C707A C707A

3 3 3

Pro236Gln Pro236Gln Pro236Gln

D8C D8C D8C

EGF, epidermal growth factor-like; D8C, domain of eight cysteines. a Nucleotide numbers refer to UMOD cDNA.

(Fig. 3d). The decreased M–P ratio and less secreted uromodulin into supernatant indicated that the three UMOD mutations influence uromodulin intracellular trafficking and modification. To evaluate whether deficient trafficking of mutant uromodulin was due to ER retention, we further observed intracellular distribution of mutant and wild type uromodulins by immunofluorescence staining in transiently transfected HEK293 cells. In wild type transfected cells, intracellular uromodulin localized both in the Golgi apparatus and ER as shown by colocalization with Giantin or Calnexin (Fig. 4a and b). However mutant uromodulins presented an intracellular reticular-like staining that mainly colocalized with calreticulin (Fig. 4a and b). We analyzed the frequency of colocalization of UMOD and ER/Golgi marker proteins in wild as well as mutant types of UMOD. It showed that colocalization of UMOD and ER maker protein was higher in mutant type compared with wild type. But colocalization of UMOD and Golgi marker protein was obviously low in mutant type (Table 3). This difference indicated that all the three UMOD mutations resulted in defective trafficking of mutant uromodulin protein due to ER retention. Besides this common influence, Cys248Trp and Pro236Gln mutant isoforms showed a more ER retention and less Golgi localization compared with Val109Glu mutant isoforms (Table 3).

3.4. Distribution of uromodulin in the kidney The expression of uromodulin was investigated in the kidney has proven UMOD mutations (F1-II-2) and normal kidneys. In the normal human kidney (Fig. 5a,b), uromodulin is diffusely distributed in the ascending limb of the loop of Henle and with maximal intensity on the apical membranes. The expression and staining pattern for uromodulin were significantly modified in the kidney of the patient (Fig. 5c, d). At higher magnification, the intense staining for uromodulin was diffusely intracellular, losing the characteristic apical membrane reactivity.

M. Liu et al. / Gene 531 (2013) 363–369

367

Fig. 3. Uromodulin expression in cell lysate and cell culture supernatant of transfected HEK293. a. Western blot was used to detect the expression of uromodulin in cell lysate of wild type and mutant types of uromodulin. Uromodulin showed two isoforms. The mature type (M) was 120-kDa and the precursor (P) was 98-kDa (plus the size of GFP). b. Densitometric analysis of western blot of wild type and mutant uromodulin bands. Data are presented by mean +/− SEM. M–P ratio (mature/precursor uromodulin) was obviously higher in cells transfected with wild type uromodulin vectors (1.41 +/− 0.34) than in cells transfected with Val109Glu (0.91 +/− 0.27), Cys248Trp (0.81 +/− 0.25) and Pro236Gln vectors (0.85 +/− 0.08). c. Western blot was used to detect the expression of uromodulin in cell culture supernatant of wild type and mutant types of uromodulin. d. Densitometric analysis of western blot of wild type and mutant uromodulin bands. Data are presented by mean +/− SEM. Uromodulin secreted to the supernatant was obviously higher in cells transfected with wild type uromodulin vectors (99.13 +/− 0.13) than in cells transfected with Val109Glu (52.31 +/− 0.15), Cys248Trp (50.08 +/− 0.10) and Pro236Gln vectors (50.89 +/− 0.13).

4. Discussion Here we identified two novel (Val109Glu, Cys248Trp) and one previously reported (Pro236Gln) (Wolf et al., 2003, 2007) UMOD mutations in the three FJHN families. We then constructed mutant uromodulin vectors of Val109Glu, Pro236Gln, and Cys248Trp by site mutagenesis

from wild type uromodulin-GFP vectors, and transfected them into HEK293 cells. Western blot of cell lysate and supernatant demonstrated that the ratio of mature over immature uromodulin (M–P ratio) was obviously higher in wild type than mutant type transfected cells. Furthermore, mutant types of uromodulin secreted into the supernatant medium were much less compared with wild type. We also identified

Fig. 4. Immunofluorescence staining indicates intracellular location of uromodulin in transiently transfected HEK293 cells. Intracellular distribution of wild-type (WT) and mutant uromodulin proteins expressed in HEK293 cells was presented. Cells transfected with GFP-tagged WT, Val109Glu, Cys248Trp or Pro236Gln were costained with anti-Calnexin—ER antibodies and anti-Giantian—Golgi antibodies.

368

M. Liu et al. / Gene 531 (2013) 363–369

Table 3 Quantitative scoring of frequency of colocalization of UMOD and ER/Golgi marker proteins. UMOD vector

Frequency

Types

Golgi + uromodulin/uromodulin

ER + uromodulin/uromodulin

WT Val109Glu Cys248Trp Pro236Gln P value

61.1% 40% 18.2% 26.7% 8.86 ∗ 10−10

55% 76.7% 86.2% 82.6% 6.31 ∗ 10−10

abnormal intracellular distribution of mutant uromodulins by immunofluorescence staining in transiently transfected HEK293 cells. The three types of mutant uromodulins presented more in ER and less in Golgi, suggesting defective trafficking of the mutant uromodulin proteins. Phenotype screening in family F1 found that two elder individuals carrying the UMOD Val109Glu mutations are still absent of clinical features of hyperuricemia or decreased glomerular filtration rate (GFR). Although we did not identify the same variation in our normal controls, the number of normal controls we choose was not enough to identify rare variations. Totally 67 missense variations in coding region of UMOD were identified according to the public database (http://www. ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?geneId=7369). Two variations with very low minor allele frequency were confirmed around position 109, one was Thr107Ala (MAF 0.001), and the other one was Glu111Glu. So there is still possibility that Val109Glu is a very rare SNP. However, with rapid increase of sequencing data, more and more rare variations were identified. Rare SNP still can influence protein function and increase susceptibility to disease. Although all of the three mutant uromodulins showed defect trafficking compared with the wild type, functional analyses revealed differences in the protein behaviors. The frequency of colocalization of UMOD and ER/Golgi marker indicated that Val109Glu protein may not be a fully functional defect compared with the other two mutant types. This phenomenon indicated that

there was less uromodulin excretion in persons carrying Val109Glu variation, which was in some circumstance not enough to keep the function of uromodulin and finally result in disease, but in some persons showed incomplete penetrance. In the family F3, one mutation Pro236Gln carrier does not have any signs of hyperuricemia and renal insufficiency, but he is younger than the proband. Whether he will proceed to disease status still needs to be followed. Similar cases were also described in other reports (Smith et al., 2011; Wolf et al., 2007). In this study, we found one reported mutation Cys248Trp in family F2. This mutation has been identified in five kindreds from Europe and Turkey (Wolf et al., 2003, 2007). Thus we assume that Cys248Trp mutation was a common mutation of UMOD in FJHN/MCKD patients. Mutations in UMOD affect biosynthesis of the protein, which leads to aberrant intracellular trafficking of uromodulin, ER storage of the protein, altered formation of supramolecular domains on the apical plasma membrane, and finally abnormal uromodulin expression in the kidney and decreased urinary uromodulin excretion (Benetti et al., 2009; Bernascone et al., 2006; Choi et al., 2005; Jennings et al., 2007; Smith et al., 2011; Vyletal et al., 2006; Williams et al., 2009). The mutation Val109Glu is located in cbEGF3, which is one of the residues coordinated with Ca2+-binding site. The cbEGF domain is also strongly recognized by ER chaperones (Whiteman and Handford, 2003). Mutation in this domain will lead to misfolded proteins and ER retention. Mutation Cys248Trp and Pro236Gln are both located within the D8C. The function of the D8C is unknown. It consists of eight conserved cysteine residues that are probably involved in disulfide bond formation (Yang et al., 2004). Previous study indicated that the Cys248Trp mutation could lead to decreased ciliary uromodulin expression in patients and transfected cells (Zaucke et al., 2010). Here we identified that mutation Cys248Trp can delay uromodulin maturation and results in protein retention in ER. Pro236Gln is another novel UMOD mutation, which leads to trafficking defect of uromodulin in our study. Other types of amino acid replacements, such as Pro236Leu and Pro236Arg

Fig. 5. Immunohistochemical analysis of uromodulin expression in the kidney. In the normal kidney, (a, b) uromodulin shows diffuse cytoplasmic staining with maximal intensity on the apical membranes. (c, d) The renal staining for uromodulin in familial juvenile hyperuricemic nephropathy (FJHN) patient (F1-II-2) was significantly modified, losing the characteristic apical membrane reactivity.

M. Liu et al. / Gene 531 (2013) 363–369

(Bernascone et al., 2006; Kudo et al., 2004) at the same position were found in different families, suggesting that the position Pro236 was a hot mutation site. Misfolded uromodulin protein retard in endoplasmic reticulum, could activate the stress signaling pathway, then generate inflammatory responses mediated by NF-κB (nuclear factor-κB) and JNK (c-Jun N-terminal kinase) activation, and finally result in progressive tissue damage (Ron and Walter, 2007; Zhang and Kaufman, 2004, 2008). In this study, the vector we used is a GFP C-terminal fusion protein. Generally N-terminal tagging vector is more likely to influence secretion of the protein comparing with C-terminal tagging vector. Uromodulin is a GPI-anchored protein, C-terminal tagging probably affects GPI-anchoring or leading to cleavage between GFP and UMOD. So we compared effects of uromodulin express vector and C-terminal tagging uromodulin vector on uromodulin trafficking by western blot. The ratio of mature over immature uromodulin (M–P ratio) was obviously higher in cells transfected with wild type uromodulin vectors than in cells transfected with mutant types, which was similar with C-terminal tagging vector. 5. Conclusions We have identified three UMOD mutations in Chinese patients with FJHN, two novels and one previously reported. We also demonstrated that all these UMOD mutants influenced uromodulin trafficking, thus lead to ER retention and less secreted uromodulin. One of the UMOD mutations, Val109Glu presented incomplete penetrance in family members, and functional study revealed that Val109Glu protein may not be a fully functional defect compared with the other two mutant types. Conflict of interest None. Acknowledgments We are grateful to the volunteer families and individuals who participated in this study. The work was supported by the National Natural Science Foundation of China (81270820). References Benetti, E., et al., 2009. Immature renal structures associated with a novel UMOD sequence variant. Am. J. Kidney Dis. 53, 327. Bernascone, I., et al., 2006. Defective intracellular trafficking of uromodulin mutant isoforms. Traffic 7, 1567. Bollee, G., et al., 2011. Phenotype and outcome in hereditary tubulointerstitial nephritis secondary to UMOD mutations. Clin. J. Am. Soc. Nephrol. 6, 2429. Choi, S.W., Ryu, O.H., Choi, S.J., Song, I.S., Bleyer, A.J., Hart, T.C., 2005. Mutant Tamm– Horsfall glycoprotein accumulation in endoplasmic reticulum induces apoptosis reversed by colchicine and sodium 4-phenylbutyrate. J. Am. Soc. Nephrol. 16, 3006. Dahan, K., et al., 2003. A cluster of mutations in the UMOD gene causes familial juvenile hyperuricemic nephropathy with abnormal expression of uromodulin. J. Am. Soc. Nephrol. 14, 2883. Duncan, H., Dixon, A.S., 1960. Gout, familial hypericaemia, and renal disease. Q. J. Med. 29, 127.

369

Fukuoka, S., Kobayashi, K., 2001. Analysis of the C-terminal structure of urinary Tamm– Horsfall protein reveals that the release of the glycosyl phosphatidylinositolanchored counterpart from the kidney occurs by phenylalanine-specific proteolysis. Biochem. Biophys. Res. Commun. 289, 1044. Hart, T.C., et al., 2002. Mutations of the UMOD gene are responsible for medullary cystic kidney disease 2 and familial juvenile hyperuricaemic nephropathy. J. Med. Genet. 39, 882. Jennings, P., et al., 2007. Membrane targeting and secretion of mutant uromodulin in familial juvenile hyperuricemic nephropathy. J. Am. Soc. Nephrol. 18, 264. Kudo, E., et al., 2004. Familial juvenile hyperuricemic nephropathy: detection of mutations in the uromodulin gene in five Japanese families. Kidney Int. 65, 1589. Kumar, S., Muchmore, A., 1990. Tamm–Horsfall protein—uromodulin (1950–1990). Kidney Int. 37, 1395. Lee, D.H., Kim, J.K., Oh, S.E., Noh, J.W., Lee, Y.K., 2010. A case of familial juvenile hyperuricemic nephropathy with novel uromodulin gene mutation, a novel heterozygous missense mutation in Korea. J. Korean Med. Sci. 25, 1680. Miller, S.A., Dykes, D.D., Polesky, H.F., 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16, 1215. Nakayama, M., et al., 2012. A Japanese family suffering from familial juvenile hyperuricemic nephropathy due to a rare mutation of the uromodulin gene. Case Rep. Nephrol. Urol. 2, 15. Ron, D., Walter, P., 2007. Signal integration in the endoplasmic reticulum unfolded protein response. Nat. Rev. Mol. Cell Biol. 8, 519. Santambrogio, S., et al., 2008. Urinary uromodulin carries an intact ZP domain generated by a conserved C-terminal proteolytic cleavage. Biochem. Biophys. Res. Commun. 370, 410. Scolari, F., et al., 1999. Identification of a new locus for medullary cystic disease, on chromosome 16p12. Am. J. Hum. Genet. 64, 1655. Serafini-Cessi, F., Malagolini, N., Hoops, T.C., Rindler, M.J., 1993. Biosynthesis and oligosaccharide processing of human Tamm–Horsfall glycoprotein permanently expressed in HeLa cells. Biochem. Biophys. Res. Commun. 194, 784. Serafini-Cessi, F., Malagolini, N., Cavallone, D., 2003. Tamm–Horsfall glycoprotein: biology and clinical relevance. Am. J. Kidney Dis. 42, 658. Smith, G.D., et al., 2011. Characterization of a recurrent in-frame UMOD indel mutation causing late-onset autosomal dominant end-stage renal failure. Clin. J. Am. Soc. Nephrol. 6, 2766. van Rooijen, J.J., Voskamp, A.F., Kamerling, J.P., Vliegenthart, J.F., 1999. Glycosylation sites and site-specific glycosylation in human Tamm–Horsfall glycoprotein. Glycobiology 9, 21. Vyletal, P., et al., 2006. Alterations of uromodulin biology: a common denominator of the genetically heterogeneous FJHN/MCKD syndrome. Kidney Int. 70, 1155. Vyletal, P., Bleyer, A.J., Kmoch, S., 2010. Uromodulin biology and pathophysiology—an update. Kidney Blood Press. Res. 33, 456. Wei, X., et al., 2012. Novel uromodulin mutation in familial juvenile hyperuricemic nephropathy. Am. J. Nephrol. 36, 114. Whiteman, P., Handford, P.A., 2003. Defective secretion of recombinant fragments of fibrillin-1: implications of protein misfolding for the pathogenesis of Marfan syndrome and related disorders. Hum. Mol. Genet. 12, 727. Williams, S.E., et al., 2009. Uromodulin mutations causing familial juvenile hyperuricaemic nephropathy lead to protein maturation defects and retention in the endoplasmic reticulum. Hum. Mol. Genet. 18, 2963. Wolf, M.T., et al., 2003. Mutations of the uromodulin gene in MCKD type 2 patients cluster in exon 4, which encodes three EGF-like domains. Kidney Int. 64, 1580. Wolf, M.T., et al., 2007. The uromodulin C744G mutation causes MCKD2 and FJHN in children and adults and may be due to a possible founder effect. Kidney Int. 71, 574. Yang, H., Wu, C., Zhao, S., Guo, J., 2004. Identification and characterization of D8C, a novel domain present in liver-specific LZP, uromodulin and glycoprotein 2, mutated in familial juvenile hyperuricaemic nephropathy. FEBS Lett. 578, 236. Zaucke, F., et al., 2010. Uromodulin is expressed in renal primary cilia and UMOD mutations result in decreased ciliary uromodulin expression. Hum. Mol. Genet. 19, 1985. Zhang, K., Kaufman, R.J., 2004. Signaling the unfolded protein response from the endoplasmic reticulum. J. Biol. Chem. 279, 25935. Zhang, K., Kaufman, R.J., 2008. From endoplasmic-reticulum stress to the inflammatory response. Nature 454, 455.