Mutations in the uromodulin gene decrease urinary excretion of Tamm-Horsfall protein

Kidney International, Vol. 66 (2004), pp. 974–977

Mutations in the uromodulin gene decrease urinary excretion of Tamm-Horsfall protein ANTHONY J. BLEYER, THOMAS C. HART, ZAK SHIHABI, VICKI ROBINS, and JOHN R. HOYER Section on Nephrology, Wake Forest University School of Medicine Medical Center, Winston-Salem, North Carolina; Department of Human Genetics, Graduate School of Public Health University of Pittsburgh, Pittsburgh, Pennslyvania; Human and Craniofacial Genetics Section, National Institute of Dental and Craniofacial Research, and National Institutes of Health, Bethesda, Maryland; Department of Pathology, Wake Forest University School of Medicine, Medical Center, Winston-Salem, North Carolina; and Department of Pediatrics and Department of Medicine, University of Pennsylvania and Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania

tic kidney disease type 2 (MCKD 2) or as uromodulin (UMOD)-associated kidney disease leads to precocious gout and progressive chronic renal failure. Genetic analysis has recently shown that mutations in the UMOD gene that encodes Tamm-Horsfall protein (THP) result in this autosomal-dominant condition [1–5]. THP is produced exclusively in the thick ascending limb of Henle’s loop (TAL) and early distal convoluted tubule [6]. This surface membrane protein is the most abundant protein in normal urine [7]. Urinary THP excretion has been evaluated by either 24-hour excretion [7–9] or in relation to creatinine excretion [9, 10]. Urinary THP decreases with declining glomerular filtration rate (GFR) [9, 10]. Since the ratio of urinary THP to creatinine is stable after 4 years of age [10], the THP excretion of adults and children can be compared. Although THP had been initially described nearly 4 decades earlier [11], the nucleotide sequence encoding THP was determined during investigations of a subspecies of this protein isolated from the urine of pregnant women. Since this protein had been called uromodulin [12], the nucleotide sequence was designated as the UMOD gene. In the present studies, we have investigated the effect of mutations in the UMOD gene on protein expression by quantitatively determining THP excretion. A sensitive enzyme-linked immunosorbent assay (ELISA) was used to measure the excretion of THP by members of an extended family that share a deletion in the UMOD gene, unaffected individuals in this family, and a smaller number of patients with other mutations of the UMOD gene.

Mutations in the uromodulin gene decrease urinary excretion of Tamm-Horsfall protein. Background. Mutations in the uromodulin (UMOD) gene that encodes Tamm-Horsfall protein (THP) cause an autosomal-dominant form of chronic renal failure. We have now investigated effects of UMOD gene mutations on protein expression by quantitatively measuring THP excretion. Methods. THP excretion was determined by enzyme-linked immunosorbent assay (ELISA) of urine collections obtained from 16 related individuals with a 27 bp deletion in the UMOD gene and seven individuals with other UMOD mutations. THP excretion of 22 control subjects (18 genetically related individuals and four spouses in the UMOD deletion family) was also determined. Results. The 16 individuals carrying the deletion mutation excreted 5.8 ± 6.3 mg THP/g creatinine into their urine. The 18 unaffected relatives from the same family excreted 40.8 ± 9.7 mg THP/g creatinine (P < 0.0001) and the four spouses excreted 43.9 ± 25.1 mg THP/g creatinine (P < 0.0001 vs. individuals with the deletion mutation). THP excretion of sevem individuals with other UMOD gene mutations was also extremely low (range of 0.14 to 5.9 mg THP/g creatinine). All individuals with UMOD mutations had low THP excretion, irrespective of gender, glomerular filtration rate (GFR), or age. Conclusion. These studies quantitatively show that the autosomal-dominant gene mutations responsible for UMODassociated kidney disease cause a profound reduction of THP excretion. We speculate that this suppression of normal THP excretion reflects deleterious effects of mutated THP within the kidney. Such effects may also play an important role in the pathogenesis of the progressive renal failure observed in patients with UMOD gene mutations.

The clinical condition known as either familial juvenile hyperuricemic nephropathy (FJHN) or medullary cysKey words: Tamm-Horsfall protein, familial juvenile hyperuricemic nephropathy, medullary cystic kidney disease, uromodulin-associated kidney disease, human.

METHODS All participants provided informed consent to a study protocol approved by the Institutional Review Board of Wake Forest University School of Medicine. Urine and blood samples were obtained from individuals from five families with known disease-associated mutations in

Received for publication January 9, 2004 and in revised form March 17, 2004, and April 13, 2004 Accepted for publication April 21, 2004
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Bleyer et al: Mutations decrease Tamm-Horsfall protein excretion

Table 1. Features of families with mutations in the gene that encodes Tamm-Horsfall protein (THP) Family

Mutation

1 (N = 16) 2

g.1966 1922deletion g.1990C>G

3

g.1880G>A

4 5 1 (unaffected)

g.2086T>C g.2105G>A

1 (spouses) (N = 4) a b

Age years

Serum creatinine

Urinary THP/ creatinine ratio

Urinary THP mg/24 hours

37.9 ± 14.2 16.4 35.5 10.9 23.6 35.7 17.0 51.0 29.2 ± 17.2 (N = 18) 47.5 ± 8.3

1.85 ± 0.93 1.3 2.2 0.8 2.0 2.2 1.1 2.0 0.95 ± 0.13 (N = 17) 1.05 ± 0.17

6.0 ± 6.1 2.5 0.56 5.9 4.4 0.14 5.6 1.3 40.8 ± 9.7a (N = 18) 43.9 ± 25.1a

5.5 ± 5.3 6.5 0.53 3.0 4.5 0.11 3.2 1.3 43.8 ± 27.9a (N = 18)b 55.4 ± 36.8a

P < 0.0001 vs. family 1 with deletion mutation. Range of 21 to 119 mg THP/24 hours.

the UMOD gene. They collected 24-hour urinary samples while on an ad libitum diet. Each urine sample was preserved with 1 mg/mL thimerosal (Sigma-Aldrich, St. Louis, MO, USA) and 0.02% sodium azide (Fisher Scientific, Hampton, NH, USA). Serum samples for uric acid and creatinine were also obtained. The creatinine measurements were performed by the Jaffe´ alkaline picrate kinetic method [13]. The uric acid measurements were performed on the ADVIA 1650 Chemistry System (Bayer Diagnostics, Tarrytown, NY, USA). This uric acid method is based on the Fossati enzymatic reaction using uricase with a Trinder-like end point [14].

clonal rabbit anti-THP prepared as previously described [17], and the secondary antibody was an affinity purified peroxidase-conjugated goat antirabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA). Concentrations of THP in samples were calculated using the curve derived from the optical densities of THP standards run on the same plate. The THP level of the urine sample from one subject was below the detection limit for the assay (∼1 ng/mL after dilution) and was assigned a value of 0.01 lg/mL for calculations and graphing. All results of groups are expressed as mean ± 1 SD. Potential differences between groups were evaluated by the unpaired Student t test.

Genetic studies Peripheral venous blood samples were obtained by standard venipuncture and genomic DNA was isolated using the QIAmp blood kit (Qiagen, Valencia, CA, USA). Polymerase chain reaction (PCR) amplification of the UMOD gene was performed using oligonucleotide primers [1]. Amplified DNA was purified with the QIAquick PCR Purification Kit (Qiagen) and was sequenced using the BigDye Terminator Cycle Sequencing Kit on an ABI 3700 DNA Analyzer (Applied Biosystems, Foster City, CA, USA) by the Genomics and Proteomics Core Laboratories of the University of Pittsburgh. Sequence analysis was performed with Sequencher 4.1 software (GeneCodes, Ann Arbor, MI, USA).

RESULTS Urine and blood samples were obtained from the 45 individuals listed in Table 1. Gene sequence analysis showed that 16 individuals from a very large family (family 1) had a 27 bp deletion (g.1966 1922del) in exon 4 of the UMOD gene. The clinical characteristics of this family have been extensively described [18]. The affected individuals from four additional families had single nucleotide mutations in exon 4 of the UMOD gene as follows: three individuals had a g.1990C>G mutation (family 2), two individuals had a g.1880G>A mutation (family 3), one individual had a g.2086T>C mutation [1] (family 4), and one individual had a g.2105G>A mutation (family 5). Sequence analysis of the 18 additional genetically related individuals in family 1 in the present study revealed no mutations in exon 4 of the UMOD gene. Analysis of samples from four genetically unrelated family 1 spouses provided additional control observations. The urinary THP excretion of affected members of family 1 was markedly lower than that of unaffected family members (Table 1). The difference between groups within this kindred was highly significant (P < 0.0001) for comparisons of either the ratio of milligram THP to gram urinary creatinine or milligram THP per 24 hours. Differences in the level of THP excretion by affected

Immunoassay The ELISA for THP was performed using a general protocol previously described in detail for human urinary osteopontin [15, 16] with the following modifications for THP. Urine samples were diluted with a 0.01 mol/L PO4, pH 9.3, buffer prior to coating of 96-well Maxisorb microtiter plates (Nunc, Rochester, NY, USA) using a series of twofold dilutions after an initial dilution of urine at 1:100 or 1:400. The primary antibody was a poly-

Urinary THP/creatinine ratio

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Bleyer et al: Mutations decrease Tamm-Horsfall protein excretion

F#1 mutation F#2-#5 mutation

80 60

F#1 unaffected F#1 spouse

40 20 0 0.0

1.0 2.0 3.0 Serum creatinine, mg/dL

4.0

Urinary THP/creatinine ratio

Fig. 1. Comparison of urinary Tamm-Horsfall protein (THP)/creatinine ratios to the serum creatinine levels of individuals with a mutation in the gene encoding THP in family 1 and in families 2 to 5 with values of the unaffected individuals in family 1 and family 1 spouses.

80 60 40 20 0 0

10

20

30 40 Age, years

50

60

70

Fig. 2. Comparison of urinary Tamm-Horsfall protein (THP)/creatinine ratios to the age of the individuals in the same groups as in Figure 1 using the same set of symbols.

individuals and the spouses in family 1 were also highly significant (P < 0.0001 for both comparisons). Many affected members of family 1 had higher serum creatinine levels, a finding consistent with their underlying disorder. The GFR of patients with UMOD gene mutations usually begins to decline in the late teens. THP excretion of affected individuals was lowest at high serum creatinine levels (Fig. 1). However, it is noteworthy that the three affected family 1 members with the lowest serum creatinine levels (0.8 mg/dL) (the GFR estimated for these three individuals using the four variable modified MDRD equation [abstract; Levey AS et al, J Am Soc Nephrol 11:155A, 2000] was 98 ± 6.0 mL/min/ 1.73 m2 ) had very low THP excretion in comparison to that of unaffected family members. The urinary THP/creatinine ratio for these three individuals was 15.3 ± 2.3 (P = 0.0003 vs. the 18 unaffected family members). The relationship of THP excretion to age is shown in Figure 2. Affected individuals had low urinary THP/creatinine ratios at all ages.

DISCUSSION All 16 members of family 1 sharing a UMOD gene mutation had lower urinary excretion of THP than any family members without the mutation (Figs. 1 and 2) (Table 1). The urinary excretion of THP was low, even at the earliest ages before developing chronic renal failure. Very low urinary THP excretion was also observed in all seven individuals with other mutations in exon 4 of the UMOD gene (Table 1) (Figs. 1 and 2) regardless of age, gender, or level of renal function. The present quantitative evidence for markedly decreased THP excretion by patients with mutations in exon 4 of the UMOD gene is consistent with and extends recent reports [4, 19]. These studies described decreased THP on Western blots of urine from a small number of patients with single nucleotide mutations of the UMOD gene [4, 19]. Decreased THP excretion appears to be a general feature of chronic renal failure [9, 10]. However, Dahan et al [4] recently reported that THP detected on Western blots of urine from patients with creatinine clearances less than 40 mL/min was decreased in patients with UMOD mutations when compared to THP of patients with other causes of renal failure. Our studies early in the disease provide additional evidence that renal failure is not the primary factor causing the markedly decreased THP excretion by patients with UMOD mutations. A precise correlation of markedly decreased THP excretion with UMOD mutations detected by genetic testing was demonstrated in the present study. This suggests that, in the future, tests of THP excretion may provide a noninvasive way to clinically evaluate young children in families with known UMOD mutations. However, DNA testing is now available (Athena Diagnostics, Worcester, MA, USA), while measurements of THP excretion are now performed only in a research setting. Thus, determining THP excretion is not currently a practical screening test for the relatively small number of individuals in families with this disease. The 616 amino acid sequence of normal THP contains an abundance of domains with an extremely high cysteine content. Such cysteine residues are believed to be important determinants of the quaternary structure of normal THP via intrachain disulfide bonds [20]. It is thus noteworthy that most of the described mutations in the UMOD gene have occurred in the exons encoding cysteine-rich domains. Indeed, most of these mutations have involved a cysteine substitution [1]. The formation of disulfide bonds in the endoplasmic reticulum may be critical for the subsequent export of THP from TAL cells [21]. This concept is supported by the recent observation of THP deposits in the endoplasmic reticulum in TAL cells in kidneys of individuals with UMOD gene mutations [4, 19].

Bleyer et al: Mutations decrease Tamm-Horsfall protein excretion

Our studies expand the types of UMOD gene mutations influencing THP excretion by showing that not only missense mutations, but also deletions markedly decrease THP excretion. The mutation in family 1 (g.1966 1922del) causes deletion of 9 amino acids from the THP peptide sequence. Single copy mutations that completely block production of a protein (null mutation) would not be expected to influence secretion of the protein encoded by the other (normal) allele. Thus, a null mutation of the UMOD gene would be expected to reduce THP excretion to half of normal. However, our quantitative studies of individuals with this deletion mutation showed that levels of excretion were much less than half the amount of THP excreted by their unaffected relatives. Therefore, it is likely that excretion of normal THP molecules is suppressed by presence of the mutated protein. Since the decrease of THP excretion occurs early when the GFR is normal, the decreased THP excretion is not simply due to renal failure. The production of a mutated protein in this disease may have deleterious effects within the kidney that are analogous to effects found as a consequence of dominant interference caused by mutations in other diseases such as ataxia telangiectasia [22] and pseudoachondroplasia [23]. UMOD-associated kidney disease appears to be one of a growing number of endoplasmic reticulum storage diseases, in which an abnormal protein precipitates in the endoplasmic reticulum with deleterious consequences [24]. We speculate that such effects play an important role in the pathogenesis of the progressive renal failure observed in patients with UMOD gene mutations. ACKNOWLEDGMENTS These studies were supported by research grants from the National Institutes of Health DK62252 and DK33501 and by a grant from the National Kidney Foundation of North Carolina, Inc. The institutions of two of the authors (Dr. Anthony Bleyer and Dr. Thomas Hart) have a licensing agreement with Athena Diagnostics, Inc., for genetic testing of individuals suspected of suffering from uromodulin-associated kidney disease. None of the authors have any ownership interest in Athena Diagnostics, Inc. Reprint requests to Anthony J. Bleyer, M.D., Section on Nephrology, Wake Forest University School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157. E-mail: [email protected] Correspondence to John R. Hoyer, M.D., The Children’s Hospital of Philadelphia, 702 Abramson Research Building, 3615 Civic Center Boulevard, Philadelphia, PA 19104–4318. E-mail: [email protected]

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