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.     

Urinary Uromodulin Excretion Predicts Progression of Chronic Kidney Disease Resulting from IgA Nephropathy

Background

Uromodulin, or Tamm-Horsfall protein, is the most abundant urinary protein in healthy individuals. Recent studies have suggested that uromodulin may play a role in chronic kidney diseases. We examined an IgA nephropathy cohort to determine whether uromodulin plays a role in the progression of IgA nephropathy.

Methods

A total of 344 IgA nephropathy patients were involved in this study. Morphological changes were evaluated with the Oxford classification of IgA nephropathy. Enzyme Linked Immunosorbent Assay (ELISA) measured the urinary uromodulin level on the renal biopsy day. Follow up was done regularly on 185 patients. Time-average blood pressure, time-average proteinuria, estimated glomerular filtration rate (eGFR) and eGFR decline rate were caculated. Association between the urinary uromodulin level and the eGFR decline rate was analyzed with SPSS 13.0.

Results

We found that lower baseline urinary uromodulin levels (P = 0.03) and higher time-average proteinuria (P = 0.04) were risk factors for rapid eGFR decline in a follow-up subgroup of the IgA nephropathy cohort. Urinary uromodulin level was correlated with tubulointerstitial lesions (P = 0.016). Patients that had more tubular atrophy/interstitial fibrosis on the surface had lower urinary uromodulin levels (P = 0.02).

Conclusions

Urinary uromodulin level is associated with interstitial fibrosis/tubular atrophy and contributes to eGFR decline in IgA nephropathy.     

IL-2, a lectin with specificity for high mannose glycopeptides

Utilizing a solid phase binding assay, we have demonstrated that rIL-2 binds with high affinity to the human urinary glycoprotein uromodulin. This binding is specifically inhibited by the saccharides diacetylchitobiose and Man(alpha 1-3)(Man(alpha 1-6]Man-O-methyl and by the high mannose glycopeptides Man5GlcNAc2-R and Man6GlcNAc2-R, but not by Man9GlcNAc2-R. rIL-2 also binds OVA, a glycoprotein which contains approximately 50% high mannose chains at a single glycosylation site, and to yeast mannan. This binding is inhibited by the same battery of saccharides which inhibit the binding to uromodulin. The conclusion that rIL-2 is a lectin is further supported by the observation that the sequence of IL-2 shares 27% homology with a 33-residue sequence of the carbohydrate-binding domain of human mannose-binding protein. The potential physiologic relevance of the carbohydrate binding activity is further elucidated by studies which show that 1) binding of soluble rIL-2 to immobilized uromodulin is enhanced at a pH of 4 to5 in the presence of divalent cations, and 2) neither uromodulin nor the high mannose glycopeptide Man5GlcNAc2Asn blocks the binding of rIL-2 to the IL-2R. Thus the carbohydrate-binding site of rIL-2 is distinct from the cell surface receptor-binding site, and might function preferentially in acidic microenvironments.     

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.     

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.     

Native cytokines do not bind to uromodulin (Tamm-Horsfall glycoprotein)

Uromodulin bound with high affinity to human tumour necrosis factor (TNF) coated on microtitre plates. This interaction was not competitively inhibited by native TNF in solution. No interaction was observed between immobilized uromodulin and TNF in the liquid phase unless conditions were chosen which denatured the latter protein. Recombinant interleukin-1 alpha adsorbed on microtitre plates also interacted with uromodulin. However, gel filtration experiments demonstrated no interaction between the proteins in the liquid phase. These and additional results indicate that uromodulin interacts with denatured cytokines, but not with native, soluble cytokines.     

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.     

In vitro evidence that carbohydrate moieties derived from uromodulin, an 85,000 dalton immunosuppressive glycoprotein isolated from human pregnancy urine, are immunosuppressive in the absence of intact protein

Our laboratory recently reported the purification of a unique immunosuppressive glycoprotein isolated from human pregnancy urine (7). This glycoprotein, which we term uromodulin, has a m.w. of 85,000 as assessed on SDS-PAGE and is 30% carbohydrate. Uromodulin blocks in vitro antigen-specific T cell proliferation to recall antigens such as tetanus toxoid at concentrations as low as 100 pM. This glycoprotein also blocks the in vitro generation of spontaneous monocyte-mediated cytotoxicity (7, 36). Recent evidence strongly suggests that the primary action of uromodulin is to act as a specific ligand and modulator of IL 1 (10, 33). We now report additional biochemical characterization of uromodulin, and based on three independent lines of evidence, find that its immunologic activity appears to result from its glycosylation. First, measures to alter the tertiary folding of the protein backbone of uromodulin, including succinylation or reduction and carboxymethylation, fail to significantly affect its in vitro bioactivity. Second, after extensive digestion of intact uromodulin with pronase, the majority of the in vitro bioactivity can be recovered in a single carbohydrate-rich fraction. Finally, digestion with N-glycanase (N-glycosidase F-, an enzyme specific for N-asparagine-linked oligosaccharides) and subsequent purification on thin layer chromatography yields a single complex oligosaccharide that appears to be responsible for the majority of the in vitro immunosuppression mediated by uromodulin. These data suggest that uromodulin displays N-linked carbohydrate sequences capable of down-regulating antigen-specific T cell responses in vitro. It has been suggested that endogenous lectins may play an important role as recognition molecules in mammalian, as well as more primitive immune systems (23, 24). Our in vitro biologic data strongly suggest that the carbohydrate portion of uromodulin is an excellent candidate to function as a potential lectin receptor.     

Defective intracellular trafficking of uromodulin mutant isoforms

Medullary cystic kidney disease/familial juvenile hyperuricemic nephropathy (MCKD/FJHN) are autosomal dominant renal disorders characterized by tubulo-interstitial fibrosis, hyperuricemia and medullary cysts. They are caused by mutations in the gene encoding uromodulin, the most abundant protein in urine. Uromodulin (or Tamm-Horsfall protein) is a glycoprotein that is exclusively expressed by epithelial tubular cells of the thick ascending limb of Henle's loop and distal convoluted tubule. To date, 37 different uromodulin mutations have been described in patients with MCKD/FJHN. Interestingly, 60% of them involve one of the 48 conserved cysteine residues. We have previously shown that cysteine-affecting mutations could lead to partial endoplasmic reticulum (ER) retention. In this study, as a further step in understanding uromodulin biology in health and disease, we provide the first extensive study of intracellular trafficking and subcellular localization of wild-type and mutant uromodulin isoforms. We analyzed a set of 12 different uromodulin mutations that were representative of the different kind of mutations identified so far by different experimental approaches (immunofluorescence, electron microscopy, biochemistry and in vivo imaging) in transiently transfected HEK293 and Madin-Darby canine kidney cells. We assessed protein processing in the secretory pathway and could demonstrate that although to different extent, all uromodulin mutations lead to defective ER to Golgi protein transport, suggesting a common pathogenetic mechanism in MCKD/FJHN.     

Uromodulin. An immunosuppressive 85-kilodalton glycoprotein isolated from human pregnancy urine is a high affinity ligand for recombinant interleukin 1 alpha

Uromodulin is an 85-kDa immunosuppressive glycoprotein originally isolated from human pregnancy urine. It exhibits immunosuppressive activity in vitro at concentrations between 10(-9) and 10(-11) M. Recent data demonstrate that uromodulin is able to specifically inhibit in vitro assays dependent upon interleukin 1 (IL-1). We now present evidence that uromodulin is a high affinity ligand for recombinant murine IL-1 alpha. Since uromodulin has been purified to homogeneity, this should allow extensive further characterization of the mechanism of action of both uromodulin and IL-1.     

Evidence that recombinant IL 1 alpha exhibits lectin-like specificity and binds to homogeneous uromodulin via N-linked oligosaccharides

Uromodulin, a recently described immunosuppressive glycoprotein isolated from human pregnancy urine, has been shown to inhibit T cell proliferative assays dependent upon interleukin 1 (IL 1). We have also recently demonstrated that uromodulin binds specifically to IL 1. We now show that not only the biologic activity but also the binding affinity of uromodulin for recombinant IL 1 is dependent upon intact glycosylation. Furthermore, oligosaccharides isolated from pronase-digested uromodulin are immunosuppressive by themselves and are able to compete with native uromodulin for binding to IL 1. We conclude that recombinant IL 1 exhibits lectin-like specificity, and uromodulin is a biologically functional glycoprotein target of the lectin-like specificity of IL 1.     

Analysis of the C-terminal structure of urinary Tamm-Horsfall protein reveals that the release of the glycosyl phosphatidylinositol-anchored counterpart from the kidney occurs by phenylalanine-specific proteolysis.

Tamm-Horsfall protein (THP), also known as uromodulin, is a major glycoprotein synthesized in the kidney. THP is expressed on the luminal surface of the membrane with the glycosyl phosphatidylinositol (GPI) anchor and excreted in urine at a rate of 50-100 mg per day. Although THP is the most abundant urinary protein, the function of THP remains unclear. In addition, little is known about the mechanism by which large amounts of THP are actively released into the urinary fluid. In this study, we examined the C-terminal structure of highly purified THP derived from human urine. Carboxypeptidase Y efficiently degraded urinary THP, indicating that the C-terminal structure of the protein contains an amino acid residue with a free carboxyl moiety. These results are consistent with our previous finding that urinary THP does not bind anti-CRD antibody. We obtained peptides from the complete digestion of urinary THP with lysylendopeptidase. We purified the most C-terminal peptide with p-phenylene diisothiocyanate-controlled pore glass (DITC-CPG) beads. N-terminal sequence analysis indicated the peptide begins with Tyr 520 and ends between E539 and E576. Direct C-terminal amino acid sequencing of highly purified urinary THP gave a sequence of -X-(Q)-G-(R)-F, corresponding to amino acids 544-548, -S-Q-G-R-F. We therefore conclude that urinary THP is generated by a proteolytic cleavage between F548 and S549, 66 amino acids upstream of a possible GPI-anchor attachment site. Because the sequence of THP, including the cleavage site, is highly homologous to that of GP2, a GPI-anchored protein within the pancreas, and both THP and GP2 are abundantly found as soluble forms in the excreted fluids, a common mechanism may exist governing the proteolytic release of GPI-anchored membrane proteins.     

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.     

Pregnancy-associated changes in the glycosylation of tamm-horsfall glycoprotein. Expression of sialyl Lewis(x) sequences on core 2 type O-glycans derived from uromodulin

Tamm-Horsfall glycoprotein (THP) is a major glycoprotein associated with human urine that binds pro-inflammatory cytokines and also inhibits in vitro T cell proliferation induced by specific antigens. THP derived from human pregnancy urine (designated uromodulin) has previously been shown to be 13-fold more effective as an inhibitor of antigen-induced T cell proliferation than THP obtained from other sources. Structural analysis of human THP and uromodulin has for the first time revealed that these glycoproteins are O-glycosylated. THP from nonpregnant females and males expresses primarily core 1 type O-glycans terminated with either sialic acid or fucose but not the sialyl Lewis(x) epitope. By contrast, the O-glycans linked to uromodulin include unusual core 2 type glycans terminated with one, two, or three sialyl Lewis(x) sequences. The specific association of these unusual carbohydrate sequences with uromodulin could explain its enhanced immunomodulatory effects compared with THP obtained from males and nonpregnant females. Analysis of THP from one of the pregnant females 2 months postpartum showed a reversion of the O-glycan profile to that found for a non-pregnant female. These data suggest that the glycosylation state of uromodulin could be under the regulation of steroidal hormones produced during pregnancy. The significant physiological implications of these observations are discussed.     

The lectin-like interaction between recombinant tumor necrosis factor and uromodulin

The polypeptide of uromodulin, an immunosuppressive glycoprotein isolated from human urine, has been shown to be identical to that of Tamm-Horsfall glycoprotein and is synthesized exclusively in the kidney (Hession, C., Decker, J. M., Sherblom, A. P., Kumar, S. (1987) Science 237, 1479-1484). Uromodulin binds recombinant murine interleukin 1 alpha with high affinity, and this binding can be inhibited by addition of specific saccharides (Muchmore, A. V., and Decker, J. M. (1987) J. Immunol. 138, 2541-2546). We now report that uromodulin binds recombinant human tumor necrosis factor (rTNF) with high affinity. Both diacetylchitobiose and Man(alpha 1-6)(Man(alpha 1-3]-Man-O-ethyl are effective inhibitors of the binding, whereas a wide variety of other saccharides are not inhibitory. Although Tamm-Horsfall glycoprotein contains predominantly tetraantennary N-linked chains, the binding to rTNF is unaffected by removal of terminal sialic acid, galactose, and N-acetylhexosamine residues. Fractionation of a Pronase digest of uromodulin by gel filtration yields material that inhibits the binding of uromodulin to rTNF but is of lower molecular weight than the major oligosaccharide. Uromodulin does not inhibit the cytotoxic activity of rTNF as monitored by lysis of tumor cell targets but effectively protects mice from lethal challenge with lipopolysaccharide, an event that may involve lymphokine toxicity. We have previously shown that rTNF binds to sections of human kidney and is localized in the same region as uromodulin. Thus, rTNF interacts with uromodulin via carbohydrate chains that are less processed than the major tetraantennary chain, and this interaction may be critical in promoting clearance and/or reducing toxicity of TNF and other lymphokines.     

Renal Tubule-Specific Expression and Urinary Secretion of Human Growth Hormone: A Kidney-Based Transgenic Bioreactor

Tissue-specific expression of human genes and secretion of human proteins into the body fluids in transgenic animals provides an important means of manufacturing large-quantity and high-quality pharmaceuticals. The present study demonstrates using transgenic mice that a 3.0 kb promoter of the mouse Tamm-Horsfall protein (THP, or uromodulin) gene directs the specific expression of human growth hormone (hGH) gene in the kidney followed by the secretion of hGH protein into the urine. hGH expression was detected in renal tubules that actively produce the THP, that is, the ascending limb of Henle's loop and distal convoluted tubules. Up to 500 ng/ml of hGH was detected in the urine, and this level remained constant throughout the 10-month observation period. hGH was also detectable in the stomach epithelium and serum in two of the transgenic lines, suggesting position-dependent effects of the transgene and leakage of hGH from the site of synthesis into the bloodstream, respectively. These results indicate that the 3.0 kb mouse THP promoter is primarily kidney-specific and can be used to convert kidney into a bioreactor in transgenic animals to produce recombinant proteins. Given the capacity of urine production independent of age, sex and lactation, the ease of urinary protein purification, and the potentially distinct machinery for post-translational modifications in the kidney epithelial cells, the kidney-based transgenic bioreactor may offer unique opportunities for producing certain complex pharmaceuticals.     

Analysis of Uromodulin Polymerization Provides New Insights into the Mechanisms Regulating ZP Domain-mediated Protein Assembly

Uromodulin is the most abundant protein secreted in urine, in which it is found as a high-molecular-weight polymer. Polymerization occurs via its zona pellucida (ZP) domain, a conserved module shared by many extracellular eukaryotic proteins that are able to assemble into matrices. In this work, we identified two motifs in uromodulin, mapping in the linker region of the ZP domain and in between protein cleavage and glycosylphosphatidylinositol (GPI)-anchoring sites, which regulate its polymerization. Indeed, mutations in either module led to premature intracellular polymerization of a soluble uromodulin isoform, demonstrating the inhibitory role of these motifs for ZP domain-mediated protein assembly. Proteolytic cleavage separating the external motif from the mature monomer is necessary to release the inhibitory function and allow protein polymerization. Moreover, we report absent or abnormal assembly into filaments of GPI-anchored uromodulin mutated in either the internal or the external motif. This effect is due to altered processing on the plasma membrane, demonstrating that the presence of the two modules has not only an inhibitory function but also can positively regulate protein polymerization. Our data expand previous knowledge on the control of ZP domain function and suggest a common mechanism regulating polymerization of ZP domain proteins.     

Goat uromodulin promoter directs kidney-specific expression of GFP gene in transgenic mice

Background  

Uromodulin is the most abundant protein found in the urine of mammals. In an effort to utilize the uromodulin promoter in order to target recombinant proteins in the urine of transgenic animals we have cloned a goat uromodulin gene promoter fragment (GUM promoter) and used it to drive expression of GFP in the kidney of transgenic mice.     

Genome-wide association study of blood pressure extremes identifies variant near UMOD associated with hypertension

Hypertension is a heritable and major contributor to the global burden of disease. The sum of rare and common genetic variants robustly identified so far explain only 1%–2% of the population variation in BP and hypertension. This suggests the existence of more undiscovered common variants. We conducted a genome-wide association study in 1,621 hypertensive cases and 1,699 controls and follow-up validation analyses in 19,845 cases and 16,541 controls using an extreme case-control design. We identified a locus on chromosome 16 in the 5′ region of Uromodulin (UMOD; rs13333226, combined P value of 3.6×10⁻¹¹). The minor G allele is associated with a lower risk of hypertension (OR [95%CI]: 0.87 [0.84–0.91]), reduced urinary uromodulin excretion, better renal function; and each copy of the G allele is associated with a 7.7% reduction in risk of CVD events after adjusting for age, sex, BMI, and smoking status (H.R. = 0.923, 95% CI 0.860–0.991; p = 0.027). In a subset of 13,446 individuals with estimated glomerular filtration rate (eGFR) measurements, we show that rs13333226 is independently associated with hypertension (unadjusted for eGFR: 0.89 [0.83–0.96], p = 0.004; after eGFR adjustment: 0.89 [0.83–0.96], p = 0.003). In clinical functional studies, we also consistently show the minor G allele is associated with lower urinary uromodulin excretion. The exclusive expression of uromodulin in the thick portion of the ascending limb of Henle suggests a putative role of this variant in hypertension through an effect on sodium homeostasis. The newly discovered UMOD locus for hypertension has the potential to give new insights into the role of uromodulin in BP regulation and to identify novel drugable targets for reducing cardiovascular risk.