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

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.     

Development of LC-MS Method for Detection of Mutant Uromodulin Protein

Mutations in the uromodulin gene cause the autosomal disorders familial juvenile hyperuricemic nephropathy (FJHN) and medullary cystic kidney disease type 2 (MCKD2). However, methods to detect the mutant form of the uromodulin protein have not been developed. In this study, we developed a liquid chromatography-mass spectrometry (LC-MS) method for detection of the mutated uromodulin peptide (C148W). Our method can distinguish the mutant peptide, GWHWE, from wildtype peptide, GWHC*E. Using MS/MS analysis with a selected reaction monitoring (SRM) mode, peptide-specific fragment ions (m/z 714 → 381, 471, 567, and 679 for GWHWE and m/z 688 → 381, 445, 541, and 653 for GWHC*E) were detected.     

Familial Nephropathy Associated with Hyperuricemia in Spain: Our Experience with 3 Families Harbouring a UMOD Mutation

Since 1993 we have studied 5 Spanish families with familial nephropathy associated with hyperuricemia (FJHN). Among these families, 24 patients have been identified. All patients had some combination of hyperuricemia, gout, renal insufficiency, arterial hypertension, and reduced kidney size. The clinical presentation in the different families and in the members of the same family was heterogeneous. Allopurinol treatment did not appear to influence renal disease. From a clinical perspective, this syndrome is a distinctive interstitial nephropathy, inherited as an autosomal dominant trait, that progresses to renal failure and is not halted nor prevented by allopurinol therapy. In 2003, genetic linkage analysis in 3 of the 5 families showed linkage of FJHN to 16p 11.2. One family was not analyzed and one family did not show linkage to this region confirming the genetic heterogeneity of this syndrome. A mutation in UMOD gene was found in these 3 families as the cause of the FJHN. The mutations cluster in exon 4 and exon 5 and were point mutation that results in an amino acid change in the uromodulin or Tamm Horsfall protein. This fact allowed in 2004, the presymptomatic genetic diagnosis of an 8-years-old boy belonging to one of these 3 Spanish families. We conclude that in families with a history of renal failure and/or gout in which FJHN is suspected, UMOD mutation screening may enable a definite diagnosis. When a mutation is found, family members can be tested for a UMOD mutation and pre-symptomatic diagnosis may allow counseling to prevent or halt the progression to renal insufficiency.     

Familial nephropathy associated with hyperuricemia in Spain: our experience with 3 families harbouring a UMOD mutation

Since 1993 we have studied 5 Spanish families with familial nephropathy associated with hyperuricemia (FJHN). Among these families, 24 patients have been identified. All patients had some combination of hyperuricemia, gout, renal insufficiency, arterial hypertension, and reduced kidney size. The clinical presentation in the different families and in the members of the same family was heterogeneous. Allopurinol treatment did not appear to influence renal disease. From a clinical perspective, this syndrome is a distinctive interstitial nephropathy, inherited as an autosomal dominant trait, that progresses to renal failure and is not halted nor prevented by allopurinol therapy. In 2003, genetic linkage analysis in 3 of the 5 families showed linkage of FJHN to 16p 11.2. One family was not analyzed and one family did not show linkage to this region confirming the genetic heterogeneity of this syndrome. A mutation in UMOD gene was found in these 3 families as the cause of the FJHN. The mutations cluster in exon 4 and exon 5 and were point mutation that results in an amino acid change in the uromodulin or Tamm Horsfall protein. This fact allowed in 2004, the presymptomatic genetic diagnosis of an 8-years-old boy belonging to one of these 3 Spanish families. We conclude that in families with a history of renal failure and/or gout in which FJHN is suspected, UMOD mutation screening may enable a definite diagnosis. When a mutation is found, family members can be tested for a UMOD mutation and pre-symptomatic diagnosis may allow counseling to prevent or halt the progression to renal insufficiency.     

Genome-wide study of familial juvenile hyperuricaemic (gouty) nephropathy (FJHN) indicates a new locus, FJHN3, linked to chromosome 2p22.1-p21

Familial juvenile hyperuricaemic (gouty) nephropathy (FJHN), is an autosomal dominant disease associated with a reduced fractional excretion of urate, and progressive renal failure. FJHN is genetically heterogeneous and due to mutations of three genes: uromodulin (UMOD), renin (REN) and hepatocyte nuclear factor-1beta (HNF-1β) on chromosomes 16p12, 1q32.1, and 17q12, respectively. However, UMOD, REN or HNF-1β mutations are found in only approximately 45% of FJHN probands, indicating the involvement of other genetic loci in approximately 55% of probands. To identify other FJHN loci, we performed a single nucleotide polymorphism (SNP)-based genome-wide linkage analysis, in six FJHN families in whom UMOD, HNF-1β and REN mutations had been excluded. Parametric linkage analysis using a 'rare dominant' model established linkage in five of the six FJHN families, with a LOD score >+3, at 0% recombination, between FJHN and SNPs at chromosome 2p22.1-p21. Analysis of individual recombinants in two unrelated affected individuals defined a approximately 5.5 Mbp interval, flanked telomerically by SNP RS372139 and centromerically by RS896986 that contained the locus, designated FJHN3. The interval contains 28 genes, and DNA sequence analysis of the most likely candidate, solute carrier family 8 member 1 (SLC8A1), did not identify any abnormalities in the FJHN3 probands. FJHN3 is likely located within a approximately 5.5 Mbp interval on chromosome 2p22.1-p21, and identifying the genetic abnormality will help to further elucidate mechanisms predisposing to gout and renal failure.     

Production and characterization of transgenic mice harboring mutant human UMOD gene

Familial juvenile hyperuricemic nephropathy is caused by mutations in the UMOD gene encoding uromodulin. A transgenic mouse model was developed by introducing a human mutant UMOD (C148W) cDNA under control of the mouse umod promoter. Uromodulin accumulation was observed in the thick ascending limb cells in the kidney of transgenic mice. However, the urinary excretion of uromodulin in transgenic mice did not decrease and LC-MS/MS analysis indicated it was of mouse origin. Moreover, the creatinine clearance was not different between wildtype and transgenic animals. Consequently, the onset of the disease was not observed in transgenic mice until 24 weeks of age.     

Detection of Mutant Uromodulin in Transgenic Mouse Harboring a Mutant Human UMOD Gene

Uromodulin is the most abundant protein secreted in urine, and the mutated form of the uromodulin gene is associated with uromodulin-associated kidney disease (UAKD). Although uromodulin accumulates in the kidney of UAKD patients, it is unclear whether this is the wildtype or mutant form. In this study, we established a liquid chromatography (LC)-mass spectrometry/mass spectrometry (MS/MS)-based method for the detection of uromodulin mutants, using the C148W mutant as a target molecule. Membrane and cytosolic fractions of kidney samples from transgenic (Tg) mice expressing the C148W uromodulin mutant were shown to contain human uromodulin by western blotting, and mutant uromodulin with the C148W mutant sequence was observed by proteomic and selected reaction monitoring analyses. Our LC-MS/MS-based method is therefore useful for detection of mutant uromodulin that is undetectable by western blotting alone.     

Detection of mutant uromodulin in transgenic mouse harboring a mutant human UMOD gene

Uromodulin is the most abundant protein secreted in urine, and the mutated form of the uromodulin gene is associated with uromodulin-associated kidney disease (UAKD). Although uromodulin accumulates in the kidney of UAKD patients, it is unclear whether this is the wildtype or mutant form. In this study, we established a liquid chromatography (LC)-mass spectrometry/mass spectrometry (MS/MS)-based method for the detection of uromodulin mutants, using the C148W mutant as a target molecule. Membrane and cytosolic fractions of kidney samples from transgenic (Tg) mice expressing the C148W uromodulin mutant were shown to contain human uromodulin by western blotting, and mutant uromodulin with the C148W mutant sequence was observed by proteomic and selected reaction monitoring analyses. Our LC-MS/MS-based method is therefore useful for detection of mutant uromodulin that is undetectable by western blotting alone.     

Endoplasmic reticulum stress as a pro-fibrotic stimulus

Current evidence suggests a prominent role for endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) in fibrotic conditions affecting a number of internal organs, including the lungs, liver, GI tract, kidney, and heart. ER stress enhances the susceptibility of structural cells, in most cases the epithelium, to pro-fibrotic stimuli. Studies suggest that ER stress facilitates fibrotic remodeling through activation of pro-apoptotic pathways, induction of epithelial–mesenchymal transition, and promotion of inflammatory responses. While genetic mutations that lead to ER stress underlie some cases of fibrosis, including lung fibrosis secondary to mutations in surfactant protein C (SFTPC), a variety of other factors can cause ER stress. These ER stress inducing factors include metabolic abnormalities, oxidative stress, viruses, and environmental exposures. Interestingly, the ability of the ER to maintain homeostasis under stress diminishes with age, potentially contributing to the fact that fibrotic disorders increase in incidence with aging. Taken together, underlying ER stress and UPR pathways are emerging as important determinants of fibrotic remodeling in different forms of tissue fibrosis. Further work is needed to better define the mechanisms by which ER stress facilitates progressive tissue fibrosis. In addition, it remains to be seen whether targeting ER stress and the UPR could have therapeutic benefit. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.     

ER stress protects from retinal degeneration

The unfolded protein response (UPR) is a specific cellular process that allows the cell to cope with the overload of unfolded/misfolded proteins in the endoplasmic reticulum (ER). ER stress is commonly associated with degenerative pathologies, but its role in disease progression is still a matter for debate. Here, we found that mutations in the ER‐resident chaperone, neither inactivation nor afterpotential A (NinaA), lead to mild ER stress, protecting photoreceptor neurons from various death stimuli in adult Drosophila. In addition, Drosophila S2 cultured cells, when pre‐exposed to mild ER stress, are protected from H₂O₂, cycloheximide‐ or ultraviolet‐induced cell death. We show that a specific ER‐mediated signal promotes antioxidant defences and inhibits caspase‐dependent cell death. We propose that an immediate consequence of the UPR not only limits the accumulation of misfolded proteins but also protects tissues from harmful exogenous stresses.     

Interaction of uromodulin and complement factor H enhances C3b inactivation

Recent studies suggest that uromodulin plays an important role in chronic kidney diseases. It can interact with several complement components, various cytokines and immune system cells. Complement factor H (CFH), as a regulator of the complement alternative pathway, is also associated with various renal diseases. Thus, we have been suggested that uromodulin regulates complement activation by interacting with CFH during tubulointerstitial injury. We detected co‐localization of uromodulin and CFH in the renal tubules by using immunofluorescence. Next, we confirmed the binding of uromodulin with CFH in vitro and found that the affinity constant (K_D) of uromodulin binding to CFH was 4.07 × 10^?6M based on surface plasmon resonance results. The binding sites on CFH were defined as the short consensus repeat (SCR) units SCR1–4, SCR7 and SCR19–20. The uromodulin‐CFH interaction enhanced the cofactor activity of CFH for factor I‐mediated cleavage of C3b to iC3b. These results indicate that uromodulin plays a role via binding and enhancing the function of CFH.     

Medullary cystic kidney disease type 1: mutational analysis in 37 genes based on haplotype sharing

Medullary cystic kidney disease type 1 (MCKD1) is an autosomal dominant, tubulo-interstitial nephropathy that causes renal salt wasting and end-stage renal failure in the fourth to seventh decade of life. MCKD1 was localized to chromosome 1q21. We demonstrated haplotype sharing and confirmed the telomeric border by a recombination of D1S2624 in a Belgian kindred. Since the causative gene has been elusive, high resolution haplotype analysis was performed in 16 kindreds. Clinical data and blood samples of 257 individuals (including 75 affected individuals) from 26 different kindreds were collected. Within the defined critical region mutational analysis of 37 genes (374 exons) in 23 MCKD1 patients was performed. In addition, for nine kindreds RT-PCR analysis for the sequenced genes was done to screen for mutations activating cryptic splice sites. We found consistency with the haplotype sharing hypothesis in an additional nine kindreds, detecting three different haplotype subsets shared within a region of 1.19 Mb. Mutational analysis of all 37 positional candidate genes revealed sequence variations in 3 different genes, AK000210, CCT3, and SCAMP3, that were segregating in each affected kindred and were not found in 96 healthy individuals, indicating, that a single responsible gene causing MCKD1 remains elusive. This may point to involvement of different genes within the MCKD1 critical region.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Trafficking abnormality and ER stress underlie functional deficiency of hearing impairment-associated connexin-31 mutants

Hearing impairment (HI) affects 1/1000 children and over 2% of the aged population. We have previously reported that mutations in the gene encoding gap junction protein connexin-31 (Cx31) are associated with HI. The pathological mechanism of the disease mutations remains unknown. Here, we show that expression of Cx31 in the mouse inner ear is developmentally regulated with a high level in adult inner hair cells and spiral ganglion neurons that are critical for the hearing process. In transfected cells, wild type Cx31 protein (Cx31wt) forms functional gap junction at cell-cell-contacts. In contrast, two HI-associated Cx31 mutants, Cx31R180X and Cx31E183K resided primarily in the ER and Golgi-like intracellular punctate structures, respectively, and failed to mediate lucifer yellow transfer. Expression of Cx31 mutants but not Cx31wt leads to upregulation of and increased association with the ER chaperone BiP indicating ER stress induction. Together, the HI-associated Cx31 mutants are impaired in trafficking, promote ER stress, and hence lose the ability to assemble functional gap junctions. The study reveals a potential pathological mechanism of HI-associated Cx31 mutations.     

Production and Characterization of Transgenic Mice Harboring Mutant Human UMOD Gene

Familial juvenile hyperuricemic nephropathy is caused by mutations in the UMOD gene encoding uromodulin. A transgenic mouse model was developed by introducing a human mutant UMOD (C148W) cDNA under control of the mouse umod promoter. Uromodulin accumulation was observed in the thick ascending limb cells in the kidney of transgenic mice. However, the urinary excretion of uromodulin in transgenic mice did not decrease and LC-MS/MS analysis indicated it was of mouse origin. Moreover, the creatinine clearance was not different between wildtype and transgenic animals. Consequently, the onset of the disease was not observed in transgenic mice until 24 weeks of age.     

Uromodulin Exclusion List Improves Urinary Exosomal Protein Identification

Advances in mass spectrometry (MS) have encouraged interest in its deployment in urine biomarker studies, but success has been limited. Urine exosomes have been proposed as an ideal source of biomarkers for renal disease. However, the abundant urinary protein, uromodulin, cofractionates with exosomes during isolation and represents a practical contaminant that limits MS sensitivity. Uromodulin depletion has been attempted but is labor- and time-intensive and may remove important protein biomarkers. We describe the application of an exclusion list (ExL) of uromodulin-related peptide ions, coupled with high-sensitivity mass spectrometric analysis, to increase the depth of coverage of the urinary exosomal proteome. Urine exosomal protein samples from healthy volunteers were subjected to tandem MS and abundant uromodulin peptides identified. Samples were run for a second time, while excluding these uromodulin peptides from fragmentation to allow identification of peptides from lower-abundance proteins. Uromodulin exclusion was performed in addition to dynamic exclusion. Results from these two procedures revealed 222 distinct proteins from conventional analysis, compared with 254 proteins after uromodulin exclusion, of which 188 were common to both methods. By unmasking a previously unidentified protein set, adding the ExL increased overall protein identifications by 29.7% to a total of 288 proteins. A fixed ExL, used in combination with conventional methods, effectively increases the depth of urinary exosomal proteins identified by MS, reducing the need for uromodulin depletion.     

Presenilin-1 mutations downregulate the signalling pathway of the unfolded-protein response

Missense mutations in the human presenilin-1 (PS1) gene, which is found on chromosome 14, cause early-onset familial Alzheimer's disease (FAD). FAD-linked PS1 variants alter proteolytic processing of the amyloid precursor protein and cause an increase in vulnerability to apoptosis induced by various cell stresses. However, the mechanisms responsible for these phenomena are not clear. Here we report that mutations in PS1 affect the unfolded-protein response (UPR), which responds to the increased amount of unfolded proteins that accumulate in the endoplasmic reticulum (ER) under conditions that cause ER stress. PS1 mutations also lead to decreased expression of GRP78/Bip, a molecular chaperone, present in the ER, that can enable protein folding. Interestingly, GRP78 levels are reduced in the brains of Alzheimer's disease patients. The downregulation of UPR signalling by PS1 mutations is caused by disturbed function of IRE1, which is the proximal sensor of conditions in the ER lumen. Overexpression of GRP78 in neuroblastoma cells bearing PS1 mutants almost completely restores resistance to ER stress to the level of cells expressing wild-type PS1. These results show that mutations in PS1 may increase vulnerability to ER stress by altering the UPR signalling pathway.     

Norepinephrine deficiency is caused by combined abnormal mRNA processing and defective protein trafficking of dopamine beta-hydroxylase

Human norepinephrine (NE) deficiency (or dopamine β-hydroxylase (DBH) deficiency) is a rare congenital disorder of primary autonomic failure, in which neurotransmitters NE and epinephrine are undetectable. Although potential pathogenic mutations, such as a common splice donor site mutation (IVS1+2T→C) and various missense mutations, in NE deficiency patients were identified, molecular mechanisms underlying this disease remain unknown. Here, we show that the IVS1+2T→C mutation results in a non-detectable level of DBH protein production and that all three missense mutations tested lead to the DBH protein being trapped in the endoplasmic reticulum (ER). Supporting the view that mutant DBH induces an ER stress response, exogenous expression of mutant DBH dramatically induced expression of BiP, a master ER chaperone. Furthermore, we found that a pharmacological chaperone, glycerol, significantly rescued defective trafficking of mutant DBH proteins. Taken together, we propose that NE deficiency is caused by the combined abnormal processing of DBH mRNA and defective protein trafficking and that this disease could be treated by a pharmacological chaperone(s).     

CLN6, which is associated with a lysosomal storage disease, is an endoplasmic reticulum protein

The neuronal ceroid lipofuscinoses (NCLs) are severe inherited neurodegenerative disorders affecting children. In this disease, lysosomes accumulate autofluorescent storage material and there is death of neurons. Five types of NCL are caused by mutations in lysosomal proteins (CTSD, CLN1/PPT1, CLN2/TTPI, CLN3 and CLN5), and one type is caused by mutations in a protein that recycles between the ER and ERGIC (CLN8). The CLN6 gene underlying a variant of late infantile NCL (vLINCL) was recently identified. It encodes a novel 311 amino acid transmembrane protein. Antisera raised against CLN6 peptides detected a protein of 30 kDa by Western blotting of human cells, which was missing in cells from some CLN6 deficient patients. Using immunofluorescence microscopy, CLN6 was shown to reside in the endoplasmic reticulum (ER). CLN6 protein tagged with GFP at the C-terminus and expressed in HEK293 cells was also found within the ER. Investigation of the effect of five CLN6 disease mutations that affect single amino acids showed that the mutant proteins were retained in the ER. These data suggest that CLN6 is an ER resident protein, the activity of which, despite this location, must contribute to lysosomal function.