Abundant Urinary Protein Research Articles

#2062 Protein interactome and trafficking of uromodulin in HEK and MDCK cell model

Abstract Background and Aims Uromodulin (UMOD) is a kidney-specific glycoprotein and the most abundant urinary protein in healthy individuals. Genome-wide association studies have shown an association between UMOD variants, kidney diseases, and hypertension. UMOD exhibits bidirectional secretion as it releases into the renal interstitium and circulation. However, the molecular trafficking of UMOD and its interaction remains poorly defined. We aimed to establish a stable Human Embryonic Kidney (HEK293) cell line expressing human UMOD to identify the potential regulators of UMOD expression or secretion. This study will provide significant implications for conditions such as hypertension, chronic kidney diseases, acute kidney injury, or UMOD-autosomal dominant tubulointerstitial kidney disease (UMOD-ADTKD). Method A HEK-UMOD-expressing cell line was generated by introducing human UMOD transcript 1 expression and using a tetracycline-inducible expression vector (pcDNA™FRT⁄TO vector) with yellow fluorescent protein tag at the C-terminal and previously established MDCK-UMOD cell line was also used. An untargeted proteomic approach was used to identify proteins, using GFP trap co-immunoprecipitation assay followed by mass spectrometry analysis. Results We identified 1367 and 1140 UMOD interacting proteins in HEK and MDCK cell models respectively. There were 620 proteins common to both these models. Through gene ontology, we unravel the molecular mechanisms involved in the biosynthesis, folding, and transport of UMOD. We observed several proteins associated with clathrin-mediated transporting. To study the role of clathrin in UMOD transport, we treated the polarised MDCK-UMOD cells with pitstop 2. There was significantly more secretion of UMOD with pitstop 2 treatment in the apical compartment compared to untreated cells however there was no significant difference in UMOD secretion in the basolateral compartment. Conclusion These results suggested that clathrin-coated vesicles are involved in transporting and sorting UMOD. Further studies need to investigate the functional importance of these proteins in UMOD trafficking and their implications in different physiological processes.

Molecular profiling of urinary extracellular vesicles in chronic kidney disease and renal fibrosis.

Chronic kidney disease (CKD) is a long-term kidney damage caused by gradual loss of essential kidney functions. A global health issue, CKD affects up to 16% of the population worldwide. Symptoms are often not apparent in the early stages, and if left untreated, CKD can progress to end-stage kidney disease (ESKD), also known as kidney failure, when the only possible treatments are dialysis and kidney transplantation. The end point of nearly all forms of CKD is kidney fibrosis, a process of unsuccessful wound-healing of kidney tissue. Detection of kidney fibrosis, therefore, often means detection of CKD. Renal biopsy remains the best test for renal scarring, despite being intrinsically limited by its invasiveness and sampling bias. Urine is a desirable source of fibrosis biomarkers as it can be easily obtained in a non-invasive way and in large volumes. Besides, urine contains biomolecules filtered through the glomeruli, mirroring the pathological state. There is, however, a problem of highly abundant urinary proteins that can mask rare disease biomarkers. Urinary extracellular vesicles (uEVs), which originate from renal cells and carry proteins, nucleic acids, and lipids, are an attractive source of potential rare CKD biomarkers. Their cargo consists of low-abundant proteins but highly concentrated in a nanosize-volume, as well as molecules too large to be filtered from plasma. Combining molecular profiling data (protein and miRNAs) of uEVs, isolated from patients affected by various forms of CKD, this review considers the possible diagnostic and prognostic value of uEVs biomarkers and their potential application in the translation of new experimental antifibrotic therapeutics.

Application of Proteogenomics to Urine Analysis towards the Identification of Novel Biomarkers of Prostate Cancer: An Exploratory Study

Simple SummaryProstate cancer (PCa) is one of the most common cancers. Due to the limited and invasive approaches for PCa diagnosis, it is crucial to identify more accurate and non-invasive biomarkers for its detection. The aim of our study was to non-invasively uncover new protein targets for detecting PCa using a proteomics and proteogenomics approach. This work identified several dysregulated mutant protein isoforms in urine from PCa patients, some of them predicted to have a protective or an adverse role in these patients. These results are promising given urine’s non-invasive nature and offers an auspicious opportunity for research and development of PCa biomarkers.To identify new protein targets for PCa detection, first, a shotgun discovery experiment was performed to characterize the urinary proteome of PCa patients. This revealed 18 differentially abundant urinary proteins in PCa patients. Second, selected targets were clinically tested by immunoblot, and the soluble E-cadherin fragment was detected for the first time in the urine of PCa patients. Third, the proteogenome landscape of these PCa patients was characterized, revealing 1665 mutant protein isoforms. Statistical analysis revealed 6 differentially abundant mutant protein isoforms in PCa patients. Analysis of the likely effects of mutations on protein function and PPIs involving the dysregulated mutant protein isoforms suggests a protective role of mutations HSPG2*Q1062H and VASN*R161Q and an adverse role of AMBP*A286G and CD55*S162L in PCa patients. This work originally characterized the urinary proteome, focusing on the proteogenome profile of PCa patients, which is usually overlooked in the analysis of PCa and body fluids. Combined analysis of mass spectrometry data using two different software packages was performed for the first time in the context of PCa, which increased the robustness of the data analysis. The application of proteogenomics to urine proteomic analysis can be very enriching in mutation-related diseases such as cancer.

POS-434 The Protective Effect of UMOD Single Nucleotide Polymorphisms (SNPs) rs12917707 on the Risk of Kidney Failure

Common genetic variations have important roles in the development and progression of chronic kidney disease (CKD), but so far most single-nucleotide polymorphisms (SNPs) described tend to have a small effect size. Uromodulin or Tamm-Horsfall, is the most abundant urinary protein in physiological conditions. Rare mutations in its encoding gene UMOD cause a Mendelian Medullary cystic kidney disease type 2 presenting in childhood. Common variants of UMOD are implicated in GFR trait in the general population.

Development of Lateral Flow Assay for the Detection of Tamm Horsfall Protein in Urine

Tamm Horsfall Protein (THP) is the most abundant urinary protein with concentrations of 20-200 mg/day and is known to be involved in a myriad of renal pathological processes such as renal stones and inflammation. THP is produced by renal epithelial cells and is the most abundant urinary protein, which tends to aggregate at higher concentrations. Therefore, our study was aimed to facilitate the detection of THP in urine by a rapid, efficient, and optimum detection using a lateral flow assay system. To develop the assay system, THP was isolated and purified from the urine of a healthy individual and used to generate antibodies in a white male New Zealand rabbit. These anti-THP antibodies were purified from the rabbit serum using the Protein-A column and further conjugated with gold nanoparticles. Gold nanoparticles (GNPs) of the size of 21 nm were synthesized by the chemical reduction method. Conjugation of gold nanoparticles and anti-THP antibodies was optimized by varying pH and concentrations. The developed lateral flow assay was tested with purified THP, and its sensitivity was calculated as 1 mg/ml of THP. This lateral flow immunoassay can detect THP in urine and can be beneficial for the detection of a various pathological or physiological phenomenon involving urinary Tamm Horsefall Protein secretions.

Uromodulin: Roles in Health and Disease.

Uromodulin, a protein exclusively produced by the kidney, is the most abundant urinary protein in physiological conditions. Already described several decades ago, uromodulin has gained the spotlight in recent years, since the discovery that mutations in its encoding gene UMOD cause a renal Mendelian disease (autosomal dominant tubulointerstitial kidney disease) and that common polymorphisms are associated with multifactorial disorders, such as chronic kidney disease, hypertension, and cardiovascular diseases. Moreover, variations in uromodulin levels in urine and/or blood reflect kidney functioning mass and are of prognostic value for renal function, cardiovascular events, and overall mortality. The clinical relevance of uromodulin reflects its multifunctional nature, playing a role in renal ion transport and immunomodulation, in protection against urinary tract infections and renal stones, and possibly as a systemic antioxidant. Here, we discuss the multifaceted roles of this protein in kidney physiology and its translational relevance.

Uromodulin – biomarker of renal function with promising clinical application

Uromodulin (also known as Tamm-Horsfall protein) is a glycoprotein produced exclusively in the kidneys, mainly in the epithelial cells of the thick ascending limb of the loop of Henle. Under physiological conditions, it is the most abundant urinary protein. A small proportion is released into the renal interstitium and then into the blood, where it can be detected and used as a potential parameter of renal function. Uromodulin has numerous physiological roles and potential pathogenetic significance, including providing protection against urinary tract infections and the formation of urinary deposits, as well as being involved in the immunomodulatory functions and regulation of water and electrolyte balance by the kidneys. Unlike classic renal markers (such as creatinine), uromodulin levels decrease with progressive renal dysfunction. A significant advantage of this parameter is therefore the detectable changes in concentration at the early stages of development of chronic kidney disease. In addition, assessment in clinical materials, such as urine and blood, is relatively simple by immunoenzymatic methods. It is evident that the quantitative determination of uromodulin in blood serum is associated with a lower risk of laboratory error and has a better correlation with renal function. Based on previous studies, Tamm-Horsfall protein / uromodulin can be considered a valuable parameter for standard diagnostics of kidney function and renal diseases. It appears that no other marker is currently able to reflect the integrity and functional state of the renal tubules as sensitively as uromodulin. Due to the potential of this parameter, the article presents and overview of the current information available about uromodulin, as well as the available diagnostic tests and the frequency of their use in clinical practice.

Study of the relationship between urinary level of uromodulin, renal involvement and disease activity in patients with systemic lupus erythrematosus.

Systemic lupus erythematosus (SLE) is a multifactorial chronic inflammatory autoimmune connective tissue disease. Lupus nephritis (LN) is a common and serious complication of SLE which can progress to end-stage renal disease. Renal biopsy is the gold standard in the diagnosis and classification of LN, but since it is an invasive procedure, it is neither desirable nor applicable for all cases. This has led to the search for an alternative, noninvasive, site-specific, and immune process-related biomarkers. Uromodulin (Tamm-Horsfall glycoprotein) is the most abundant urinary protein expressed exclusively by the thick ascending limb cells and released into urine of healthy controls. Studies showed that it may act as a danger signaling molecule eliciting an inflammatory response following conditions that damage the nephron integrity and leading to uromodulin release into the interstitial space. This study aimed to assess uromodulin as a screening biomarker of tubulointerstitial involvement in patients with SLE and to elucidate its correlation with disease activity and progression. The study was conducted on 70 patients divided into two groups: control group (Group I) consisted of 20 apparently healthy volunteers of comparable age and sex to the patients' group, and 50 SLE patients (Group II) diagnosed according to the 2012 Systemic Lupus Collaborating Clinics (SLICC) classification criteria. Group II was further subdivided into 23 patients without manifestations of LN (Group II A) and 27 patients with manifestations of LN (Group II B). Urinary uromodulin level showed statistically significant difference among the studied groups, being lowest among the LN patients with a mean value 5.6 ± 3.4, in SLE patients without nephritis 9.9 ± 5.2 and 12.9 ± 4.6 in the control group. Urinary uromodulin also correlated positively with estimated glome- rular filtration rate. A negative correlation was found between urinary uromodulin and serum creatinine, 24 h urinary proteins and SLICC renal activity score. No statistically significant correlation was found between urinary uromo- dulin and SLE disease activity index. Thus, decreasing urinary uromodulin levels can be a marker for renal involvement and tubulo- interstitial nephritis in active SLE patients and a marker for chronic kidney disease and nephron loss in the absence of activity markers.

Selective protein enrichment in calcium oxalate stone matrix: a window to pathogenesis?

Urine proteins are thought to control calcium oxalate stone formation, but over 1000 proteins have been reported in stone matrix obscuring their relative importance. Proteins critical to stone formation should be present at increased relative abundance in stone matrix compared to urine, so quantitative protein distribution data were obtained for stone matrix compared to prior urine proteome data. Matrix proteins were isolated from eight stones (> 90% calcium oxalate content) by crystal dissolution and further purified by ultradiafiltration (> 10kDa membrane). Proteomic analyses were performed using label-free spectral counting tandem mass spectrometry, followed by stringent filtering. The average matrix proteome was compared to the average urine proteome observed in random urine samples from 25 calcium oxalate stone formers reported previously. Five proteins were prominently enriched in matrix, accounting for a mass fraction of > 30% of matrix protein, but only 3% of urine protein. Many highly abundant urinary proteins, like albumin and uromodulin, were present in matrix at reduced relative abundance compared to urine, likely indicating non-selective inclusion in matrix. Furthermore, grouping proteins by isoelectric point demonstrated that the stone matrix proteome was highly enriched in both strongly anionic (i.e., osteopontin) and strongly cationic (i.e., histone) proteins, most of which are normally found in intracellular or nuclear compartments. The fact that highly anionic and highly cationic proteins aggregate at low concentrations and these aggregates can induce crystal aggregation suggests that protein aggregation may facilitate calcium oxalate stone formation, while cell injury processes are implicated by the presence of many intracellular proteins.

Uromodulin regulates renal magnesium homeostasis through the ion channel transient receptor potential melastatin 6 (TRPM6)

Up to 15% of the population have mild to moderate chronic hypomagnesemia, which is associated with type 2 diabetes mellitus, hypertension, metabolic syndrome, and chronic kidney disease. The kidney is the key organ for magnesium homeostasis, but our understanding of renal magnesium regulation is very limited. Uromodulin (UMOD) is the most abundant urinary protein in humans, and here we report that UMOD has a role in renal magnesium homeostasis. Umod-knockout (Umod-/-) mice excreted more urinary magnesium than WT mice and displayed up-regulation of genes promoting magnesium absorption. The majority of magnesium is absorbed in the thick ascending limb. However, both mouse strains responded similarly to the diuretic agent furosemide, indicating appropriate function of the thick ascending limb in the Umod-/- mice. Magnesium absorption is fine-tuned in the distal convoluted tubule (DCT) via the apical magnesium channel transient receptor potential melastatin 6 (TRPM6). We observed decreased apical Trpm6 staining in the DCT of Umod-/- mice. Applying biotinylation assays and whole-cell patch-clamp recordings, we found that UMOD enhances TRPM6 cell-surface abundance and current density from the extracellular space. UMOD physically interacted with TRPM6 and thereby impaired dynamin-dependent TRPM6 endocytosis. WT mice fed a low-magnesium diet had an increased urinary UMOD secretion compared with the same mice on a regular diet. Our results suggest that increased urinary UMOD secretion in low-magnesium states reduces TRPM6 endocytosis and thereby up-regulates TRPM6 cell-surface abundance to defend against further urinary magnesium losses.

A novel role for Tamm-Horsfall protein (uromodulin) in the renal tubule

Tamm-Horsfall protein (THP)/uromodulin, the most abundant urinary protein, is produced in the thick ascending limb of the loop of Henle. Besides immunological functions, it has regulatory impact on ion transport and volume regulation. It had been debated whether the distal convoluted tubule is a source for THP as well. Novel results suggest that in its early portions, THP synthesis affects adaptive plasticity of the epithelium, stabilizes calcium homeostasis, and activates the thiazide-sensitive Na+,Cl--cotransporter.

Tamm-Horsfall glycoprotein engages human Siglec-9 to modulate neutrophil activation in the urinary tract.

Urinary tract infections (UTI) are a major problem in human medicine for which better understanding of native immune defenses may reveal new pathways for therapeutic intervention. Tamm-Horsfall glycoprotein (THP), the most abundant urinary protein, interacts with bacteria including uropathogenic E. coli (UPEC) as well host immune cells. In addition to its well-studied functions to antagonize bacterial colonization, we hypothesize that THP serves a critical host defense function through innate immune modulation. Using isolated human neutrophils, we found that THP binds neutrophils and that this interaction reduces reactive oxygen species generation, chemotaxis, and killing of UPEC. We discovered that THP engages the inhibitory neutrophil receptor sialic acid-binding Ig-like lectin-9 (Siglec-9), and mouse functional ortholog Siglec-E, in a manner dependent on sialic acid on its N-glycan moieties. THP-null mice have significantly more neutrophils present in the urine compared to WT mice, both with and without the presence of inflammatory stimuli. These data support THP as an important negative regulator of neutrophil activation in the urinary tract, with dual functions to counteract bacterial colonization and suppress excessive inflammation within the urinary tract.

Human Tamm-Horsfall protein, a renal specific protein, serves as a cofactor in complement 3b degradation.

Tamm-Horsfall protein (THP) is an abundant urinary protein of renal origin. We hypothesize that THP can act as an inhibitor of complement since THP binds complement 1q (C1q) of the classical complement pathway, inhibits activation of this pathway, and is important in decreasing renal ischemia-reperfusion injury (a complement-mediated condition). In this study, we began to investigate whether THP interacted with the alternate complement pathway via complement factor H (CFH). THP was shown to bind CFH using ligand blots and in an ELISA (KD of 1 × 10−6 M). Next, the ability of THP to alter CFH’s normal action as it functioned as a cofactor in complement factor I (CFI)–mediated complement 3b (C3b) degradation was investigated. Unexpectedly, control experiments in these in vitro assays suggested that THP, without added CFH, could act as a cofactor in CFI-mediated C3b degradation. This cofactor activity was present equally in THP isolated from 10 different individuals. While an ELISA demonstrated small amounts of CFH contaminating THP samples, these CFH amounts were insufficient to explain the degree of cofactor activity present in THP. An ELISA demonstrated that THP directly bound C3b (KD ~ 5 × 10−8 m), a prerequisite for a protein acting as a C3b degradation cofactor. The cofactor activity of THP likely resides in the protein portion of THP since partially deglycosylated THP still retained cofactor activity. In conclusion, THP appears to participate directly in complement inactivation by its ability to act as a cofactor for C3b degradation, thus adding support to the hypothesis that THP might act as an endogenous urinary tract inhibitor of complement.

Mutant uromodulin expression leads to altered homeostasis of the endoplasmic reticulum and activates the unfolded protein response.

Uromodulin is the most abundant urinary protein in physiological conditions. It is exclusively produced by renal epithelial cells lining the thick ascending limb of Henle’s loop (TAL) and it plays key roles in kidney function and disease. Mutations in UMOD, the gene encoding uromodulin, cause autosomal dominant tubulointerstitial kidney disease uromodulin-related (ADTKD-UMOD), characterised by hyperuricemia, gout and progressive loss of renal function. While the primary effect of UMOD mutations, retention in the endoplasmic reticulum (ER), is well established, its downstream effects are still largely unknown. To gain insight into ADTKD-UMOD pathogenesis, we performed transcriptional profiling and biochemical characterisation of cellular models (immortalised mouse TAL cells) of robust expression of wild type or mutant GFP-tagged uromodulin. In this model mutant uromodulin accumulation in the ER does not impact on cell viability and proliferation. Transcriptional profiling identified 109 genes that are differentially expressed in mutant cells relative to wild type ones. Up-regulated genes include several ER resident chaperones and protein disulphide isomerases. Consistently, pathway enrichment analysis indicates that mutant uromodulin expression affects ER function and protein homeostasis. Interestingly, mutant uromodulin expression induces the Unfolded Protein Response (UPR), and specifically the IRE1 branch, as shown by an increased splicing of XBP1. Consistent with UPR induction, we show increased interaction of mutant uromodulin with ER chaperones Bip, calnexin and PDI. Using metabolic labelling, we also demonstrate that while autophagy plays no role, mutant protein is partially degraded by the proteasome through ER-associated degradation. Our work demonstrates that ER stress could play a central role in ADTKD-UMOD pathogenesis. This sets the bases for future work to develop novel therapeutic strategies through modulation of ER homeostasis and associated protein degradation pathways.

An Empirical Approach to Signature Peptide Choice for Selected Reaction Monitoring: Quantification of Uromodulin in Urine.

Many avenues have been proposed for a seamless transition between biomarker discovery data and selected reaction monitoring (SRM) assays for biomarker validation. Unfortunately, studies with the abundant urinary protein uromodulin have shown that these methods do not converge on a consistent set of surrogate peptides for targeted mass spectrometry. As an alternative, we present an empirical peptide selection work flow for robust protein quantification. We compared the relative SRM signal intensity of 12 uromodulin-derived peptides between tryptic digests of 9 urine samples. Pairwise CVs between the 12 peptides were 0.19-0.99. We used a correlation matrix to identify peptides that reproducibly tracked the amount of uromodulin protein and selected 4 peptides with robust and highly correlated SRM signals. Absolute quantification was performed with stable isotope-labeled versions of these peptides as internal standards and a standard curve prepared from a tryptic digest of purified uromodulin. Absolute quantification of uromodulin in 40 clinical urine samples yielded interpeptide correlations of ≥0.984 and correlations of ≥0.912 with ELISA data. The SRM assays were linear over >3 orders of magnitude and had typical interdigest CVs of <10%, interinjection CVs of <7%, and intertransition CVs of <7%. Comparing the apparent abundance of a plurality of peptides derived from the same target protein makes it possible to select signature peptides that are unaffected by the unpredictable confounding factors inevitably present in biological samples.

Highly sensitive reversed-phase high-performance liquid chromatography assay for the detection of Tamm-Horsfall protein in human urine.

Tamm-Horsfall protein (also known as uromodulin) is the most abundant urinary protein in healthy individuals. Since initially characterized by Tamm and Horsfall, the amount of urinary excretion and structural mutations of Tamm-Horsfall protein is associated with kidney diseases. However, currently available assays for Tamm-Horsfall protein, which are mainly enzyme-linked immunosorbent assay-based, suffer from poor reproducibility and might give false negative results. We developed a novel, quantitative assay for Tamm-Horsfall protein using reversed-phase high-performance liquid chromatography. A precipitation pretreatment avoided urine matrix interference and excessive sample dilution. High-performance liquid chromatography optimization based on polarity allowed excellent separation of Tamm-Horsfall protein from other major urine components. Our method exhibited high precision (based on the relative standard deviations of intraday [≤2.77%] and interday [≤5.35%] repetitions). The Tamm-Horsfall protein recovery rate was 100.0-104.2%. The mean Tamm-Horsfall protein concentration in 25 healthy individuals was 31.6 ± 18.8 mg/g creatinine. There was a strong correlation between data obtained by high-performance liquid chromatography and enzyme-linked immunosorbent assay (r = 0.906), but enzyme-linked immunosorbent assay values tended to be lower than high-performance liquid chromatography values at low Tamm-Horsfall protein concentrations. The high sensitivity and reproducibility of our Tamm-Horsfall protein assay will reduce the number of false negative results of the sample compared with enzyme-linked immunosorbent assay. Moreover, our method is superior to other high-performance liquid chromatography methods, and a simple protocol will facilitate further research on the physiological role of Tamm-Horsfall protein.

Tamm‐Horsfall Protein Binds Properdin

The purpose of this study was to determine if Tamm‐Horsfall protein (THP), an abundant urinary protein that interacts with various molecules of the immune system, binds to properdin (Factor P), a positive regulator of complement activation which stabilizes the alternative pathway C3 convertase. Enzyme‐linked immunosorbent assays (ELISA) were used to evaluate the binding of soluble human properdin to immobilized human THP. In low ionic strength buffers containing magnesium and calcium, THP bound properdin strongly (KD ~ 10‐8 M). Removing divalent cations from the buffer markedly reduced the ability of THP to bind properdin. Ionic strength also was important in this interaction. Properdin binding to THP was stronger in low ionic strength (20mM NaCl) and intermediate ionic strength buffers (90mM NaCl), than in physiologic strength (154mM NaCl) buffers. By ELISA, soluble THP effectively competed with immobilized THP for binding to properdin, with 0.1µM soluble THP inhibiting 50% of 0.05µM properdin's binding to immobilized THP. Western blot analysis demonstrated that soluble THP could bind to properdin after the properdin had been separated by SDS‐PAGE and transferred to nitrocellulose paper. In conclusion, this study shows that THP binds to properdin in the presence of divalent cations. This interaction is influenced by NaCl concentration, suggesting that electrostatic interactions between the two molecules are important. Properdin appears to recognize THP in solution, as well as immobilized THP, and THP binds to soluble properdin, as well as properdin that has been partially denatured by SDS during electrophoresis and Western blotting. The functional significance of this interaction remains to be evaluated. Funded by the PNWU Research Seed Program.

Uromodulin, an emerging novel pathway for blood pressure regulation and hypertension.

Tamm–Horsfall protein was discovered in 1950 by Igor Tamm and Frank Horsfall,1 using a salt precipitation procedure to isolate a potent inhibitor of viral hemagglutination from urine. Muchmore and Decker,2 in 1985, isolated a glycoprotein (calling it uromodulin) with in vitro immunosuppressive properties from urine of pregnant women. In 1987, Pennica et al3 confirmed by cDNA analysis that uromodulin and Tamm–Horsfall protein were identical proteins. Since the initial discovery and without any clear understanding of the function of uromodulin, it was only in 2002 when Hart et al4 identified causative uromodulin mutations in a subset of families having familial juvenile hyperuricemic nephropathy and medullary cystic kidney disease type 2 that interest in uromodulin biology and function was revived. Interest in uromodulin was further revitalized by genome-wide association studies (GWASs) in 2009/2010 showing an association between common single-nucleotide polymorphisms in the upstream region of the UMOD gene with renal function and hypertension.5–8 In 2013, 2 independent groups undertaking post-GWAS functional dissection of the UMOD loci provided molecular insights into a new pathway for hypertension and sodium homeostasis involving uromodulin and opening an exciting line of investigation that could enhance our understanding of renal tubule physiology, sodium homeostasis, blood pressure (BP) regulation, and potentially lead to novel therapies for hypertension.9,10 Uromodulin is a protein exclusively expressed by epithelial cells of the thick ascending limb of Henle’s loop (TAL; Figure 1). The 640 amino-acid precursor is cotranslationally translocated into the endoplasmic reticulum (ER), extensively glycosylated, glypiated, and glycosylphosphatidylinositol anchored to the apical tubular cell membrane. From here it is released by a specific, but as yet unidentified, serine protease(s). The released protein is excreted in the urine at a rate of 20 to 100 mg/d and represents the most abundant urinary protein in …

Wild-type uromodulin prevents NFkB activation in kidney cells, while mutant uromodulin, causing FJHU nephropathy, does not

Uromodulin (Tamm-Horsfall protein) is the most abundant urinary protein in healthy individuals. Despite 60 years of research, its physiological role remains rather elusive. Familial juvenile hyperuricemic nephropathy and medullary cystic kidney disease Type 2 are autosomal dominant tubulointerstitial nephropathies characterized by gouty arthritis and progressive renal insufficiency, caused by uromodulin (UMOD) mutations. The aim of this study was to compare the cellular effects of mutant and wild-type UMOD. Wild-type UMOD cDNA was cloned from human kidney cDNA into pcDNA3 expression vector. A mutant UMOD construct, containing the previously reported mutation, V273, was created by in vitro mutagenesis. Transient and stable transfection studies were performed in human embryonic kidney cells and mouse distal convoluted tubular cells, respectively. Expression was evaluated by reverse transcription polymerase chain reaction (RT-PCR), western blot and immunofluorescence. Oligosaccharide cleavage by glycosidases was performed to characterize different forms of UMOD. Nuclear translocation of P65 and degradation of IκBα and IRAK1 in response to interleukin (IL)-1β were used to evaluate the effects of wild-type and mutant UMOD on the IL-1R-NFκB pathway. The mutant protein was shown to be retained in the endoplasmic reticulum and was not excreted to the cell medium, as opposed to the wild-type protein. NFκB activation in cells expressing mutant UMOD was similar to that of untransfected cells. In contrast, cells over-expressing wild-type UMOD showed markedly reduced NFκB activation. Our findings suggest that UMOD may have a physiologic function related to its inhibitory effect on the NFκB pathway.

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

BackgroundUromodulin, 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.MethodsA 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.ResultsWe 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).ConclusionsUrinary uromodulin level is associated with interstitial fibrosis/tubular atrophy and contributes to eGFR decline in IgA nephropathy.