An intermediate-effect size variant in UMOD confers risk for chronic kidney disease.

The kidney-specific gene UMOD encodes for uromodulin, the most abundant protein excreted in normal urine. Rare large-effect variants in UMOD cause autosomal dominant tubulointerstitial kidney disease (ADTKD), while common low-impact variants strongly associate with kidney function and the risk of chronic kidney disease (CKD) in the general population. It is unknown whether intermediate-effect variants in UMOD contribute to CKD. Here, candidate intermediate-effect UMOD variants were identified using large-population and ADTKD cohorts. Biological and phenotypical effects were investigated using cell models, in silico simulations, patient samples, and international databases and biobanks. Eight UMOD missense variants reported in ADTKD are present in the Genome Aggregation Database (gnomAD), with minor allele frequency (MAF) ranging from 10-5 to 10-3. Among them, the missense variant p.Thr62Pro is detected in ∼1/1,000 individuals of European ancestry, shows incomplete penetrance but a high genetic load in familial clusters of CKD, and is associated with kidney failure in the 100,000 Genomes Project (odds ratio [OR] = 3.99 [1.84 to 8.98]) and the UK Biobank (OR = 4.12 [1.32 to 12.85). Compared with canonical ADTKD mutations, the p.Thr62Pro carriers displayed reduced disease severity, with slower progression of CKD and an intermediate reduction of urinary uromodulin levels, in line with an intermediate trafficking defect in vitro and modest induction of endoplasmic reticulum (ER) stress. Identification of an intermediate-effect UMOD variant completes the spectrum of UMOD-associated kidney diseases and provides insights into the mechanisms of ADTKD and the genetic architecture of CKD.

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.

Cryo-EM structure of native human uromodulin, a zona pellucida module polymer.

Assembly of extracellular filaments and matrices mediating fundamental biological processes such as morphogenesis, hearing, fertilization, and antibacterial defense is driven by a ubiquitous polymerization module known as zona pellucida (ZP) "domain". Despite the conservation of this element from hydra to humans, no detailed information is available on the filamentous conformation of any ZP module protein. Here, we report a cryo-electron microscopy study of uromodulin (UMOD)/Tamm-Horsfall protein, the most abundant protein in human urine and an archetypal ZP module-containing molecule, in its mature homopolymeric state. UMOD forms a one-start helix with an unprecedented 180-degree twist between subunits enfolded by interdomain linkers that have completely reorganized as a result of propeptide dissociation. Lateral interaction between filaments in the urine generates sheets exposing a checkerboard of binding sites to capture uropathogenic bacteria, and UMOD-based models of heteromeric vertebrate egg coat filaments identify a common sperm-binding region at the interface between subunits.

Peptidomic Analysis of Urine from Youths with Early Type 1 Diabetes Reveals Novel Bioactivity of Uromodulin Peptides In Vitro.

Chronic hyperglycemia is known to disrupt the proteolytic milieu, initiating compensatory and maladaptive pathways in the diabetic kidney. Such changes in intrarenal proteolysis are captured by the urinary peptidome. To elucidate the early kidney response to chronic hyperglycemia, we conducted a peptidomic investigation into urines from otherwise healthy youths with type 1 diabetes and their non-diabetic peers using unbiased and targeted mass spectrometry-based techniques. This cross-sectional study included two separate cohorts for the discovery (n = 30) and internal validation (n = 30) of differential peptide excretion. Peptide bioactivity was predicted using PeptideRanker and subsequently verified in vitro Proteasix and the Nephroseq database were used to identify putative proteases responsible for peptide generation and examine their expression in diabetic nephropathy. A total of 6550 urinary peptides were identified in the discovery analysis. We further examined the subset of 162 peptides, which were quantified across all thirty samples. Of the 15 differentially excreted peptides (p < 0.05), seven derived from a C-terminal region (589SGSVIDQSRVLNLGPITRK607) of uromodulin, a kidney-specific protein. Increased excretion of five uromodulin peptides was replicated in the validation cohort using parallel reaction monitoring (p < 0.05). One of the validated peptides (SGSVIDQSRVLNLGPI) activated NFκB and AP-1 signaling, stimulated cytokine release, and enhanced neutrophil migration in vitro. In silico analyses highlighted several potential proteases such as hepsin, meprin A, and cathepsin B to be responsible for generating these peptides. In summary, we identified a urinary signature of uromodulin peptides associated with early type 1 diabetes before clinical manifestations of kidney disease and discovered novel bioactivity of uromodulin peptides in vitro Our present findings lay the groundwork for future studies to validate peptide excretion in larger and broader populations, to investigate the role of bioactive uromodulin peptides in high glucose conditions, and to examine proteases that cleave uromodulin.

Chemically based transmissible ER stress protocols are unsuitable to study cell-to-cell UPR transmission.

Renal epithelial cells regulate the destructive activity of macrophages and participate in the progression of kidney diseases. Critically, the Unfolded Protein Response (UPR), which is activated in renal epithelial cells in the course of kidney injury, is required for the optimal differentiation and activation of macrophages. Given that macrophages are key regulators of renal inflammation and fibrosis, we suppose that the identification of mediators that are released by renal epithelial cells under Endoplasmic Reticulum (ER) stress and transmitted to macrophages is a critical issue to address. Signals leading to a paracrine transmission of ER stress (TERS) from a donor cell to a recipient cells could be of paramount importance to understand how ER-stressed cells shape the immune microenvironment. Critically, the vast majority of studies that have examined TERS used thaspigargin as an inducer of ER stress in donor cells in cellular models. By using multiple sources of ER stress, we evaluated if human renal epithelial cells undergoing ER stress can transmit the UPR to human monocyte-derived macrophages and if such TERS can modulate the inflammatory profiles of these cells. Our results indicate that carry-over of thapsigargin is a confounding factor in chemically based TERS protocols classically used to induce ER Stress in donor cells. Hence, such protocols are not suitable to study the TERS phenomenon and to identify its mediators. In addition, the absence of TERS transmission in more physiological models of ER stress indicates that cell-to-cell UPR transmission is not a universal feature in cultured cells.

Clinical and genetic spectra of autosomal dominant tubulointerstitial kidney disease due to mutations in UMOD and MUC1.

Autosomal dominant tubulointerstitial kidney disease (ADTKD) is an increasingly recognized cause of end-stage kidney disease, primarily due to mutations in UMOD and MUC1. The lack of clinical recognition and the small size of cohorts have slowed the understanding of disease ontology and development of diagnostic algorithms. We analyzed two registries from Europe and the United States to define genetic and clinical characteristics of ADTKD-UMOD and ADTKD-MUC1 and develop a practical score to guide genetic testing. Our study encompassed 726 patients from 585 families with a presumptive diagnosis of ADTKD along with clinical, biochemical, genetic and radiologic data. Collectively, 106 different UMOD mutations were detected in 216/562 (38.4%) of families with ADTKD (303 patients), and 4 different MUC1 mutations in 72/205 (35.1%) of the families that are UMOD-negative (83 patients). The median kidney survival was significantly shorter in patients with ADTKD-MUC1 compared to ADTKD-UMOD (46 vs. 54 years, respectively), whereas the median gout-free survival was dramatically reduced in patients with ADTKD-UMOD compared to ADTKD-MUC1 (30 vs. 67 years, respectively). In contrast to patients with ADTKD-UMOD, patients with ADTKD-MUC1 had normal urinary excretion of uromodulin and distribution of uromodulin in tubular cells. A diagnostic algorithm based on a simple score coupled with urinary uromodulin measurements separated patients with ADTKD-UMOD from those with ADTKD-MUC1 with a sensitivity of 94.1%, a specificity of 74.3% and a positive predictive value of 84.2% for a UMOD mutation. Thus, ADTKD-UMOD is more frequently diagnosed than ADTKD-MUC1, ADTKD subtypes present with distinct clinical features, and a simple score coupled with urine uromodulin measurements may help prioritizing genetic testing.

A structured interdomain linker directs self-polymerization of human uromodulin.

Uromodulin (UMOD)/Tamm-Horsfall protein, the most abundant human urinary protein, plays a key role in chronic kidney diseases and is a promising therapeutic target for hypertension. Via its bipartite zona pellucida module (ZP-N/ZP-C), UMOD forms extracellular filaments that regulate kidney electrolyte balance and innate immunity, as well as protect against renal stones. Moreover, salt-dependent aggregation of UMOD filaments in the urine generates a soluble molecular net that captures uropathogenic bacteria and facilitates their clearance. Despite the functional importance of its homopolymers, no structural information is available on UMOD and how it self-assembles into filaments. Here, we report the crystal structures of polymerization regions of human UMOD and mouse ZP2, an essential sperm receptor protein that is structurally related to UMOD but forms heteropolymers. The structure of UMOD reveals that an extensive hydrophobic interface mediates ZP-N domain homodimerization. This arrangement is required for filament formation and is directed by an ordered ZP-N/ZP-C linker that is not observed in ZP2 but is conserved in the sequence of deafness/Crohn's disease-associated homopolymeric glycoproteins α-tectorin (TECTA) and glycoprotein 2 (GP2). Our data provide an example of how interdomain linker plasticity can modulate the function of structurally similar multidomain proteins. Moreover, the architecture of UMOD rationalizes numerous pathogenic mutations in both UMOD and TECTA genes.

Whole-Exome Sequencing in Adults With Chronic Kidney Disease: A Pilot Study.

Background: The utility of whole-exome sequencing (WES) for the diagnosis and management of adult-onset constitutional disorders has not been adequately studied. Genetic diagnostics may be advantageous in adults with chronic kidney disease (CKD), in whom the cause of kidney failure often remains unknown. Objective: To study the diagnostic utility of WES in a selected referral population of adults with CKD. Design: Observational cohort. Setting: A major academic medical center. Patients: 92 adults with CKD of unknown cause or familial nephropathy or hypertension. Measurements: The diagnostic yield of WES and its potential effect on clinical management. Results: Whole-exome sequencing provided a diagnosis in 22 of 92 patients (24%), including 9 probands with CKD of unknown cause and encompassing 13 distinct genetic disorders. Among these, loss-of-function mutations were identified in PARN in 2 probands with tubulointerstitial fibrosis. PARN mutations have been implicated in a short telomere syndrome characterized by lung, bone marrow, and liver fibrosis; these findings extend the phenotype of PARN mutations to renal fibrosis. In addition, review of the American College of Medical Genetics actionable genes identified a pathogenic BRCA2 mutation in a proband who was diagnosed with breast cancer on follow-up. The results affected clinical management in most identified cases, including initiation of targeted surveillance, familial screening to guide donor selection for transplantation, and changes in therapy. Limitation: The small sample size and recruitment at a tertiary care academic center limit generalizability of findings among the broader CKD population. Conclusion: Whole-exome sequencing identified diagnostic mutations in a substantial number of adults with CKD of many causes. Further study of the utility of WES in the evaluation and care of patients with CKD in additional settings is warranted. Primary Funding Source: New York State Empire Clinical Research Investigator Program, Renal Research Institute, and National Human Genome Research Institute of the National Institutes of Health.

The Uromodulin Gene Locus Shows Evidence of Pathogen Adaptation through Human Evolution.

Common variants in the UMOD gene encoding uromodulin, associated with risk of hypertension and CKD in the general population, increase UMOD expression and urinary excretion of uromodulin, causing salt-sensitive hypertension and renal lesions. To determine the effect of selective pressure on variant frequency, we investigated the allelic frequency of the lead UMOD variant rs4293393 in 156 human populations, in eight ancient human genomes, and in primate genomes. The T allele of rs4293393, associated with CKD risk, has high frequency in most modern populations and was the one detected in primate genomes. In contrast, we identified only the derived, C allele in Denisovan and Neanderthal genomes. The distribution of the UMOD ancestral allele did not follow the ancestral susceptibility model observed for variants associated with salt-sensitive hypertension. Instead, the global frequencies of the UMOD alleles significantly correlated with pathogen diversity (bacteria, helminths) and prevalence of antibiotic-resistant urinary tract infections (UTIs). The inverse correlation found between urinary levels of uromodulin and markers of UTIs in the general population substantiates the link between UMOD variants and protection against UTIs. These data strongly suggest that the UMOD ancestral allele, driving higher urinary excretion of uromodulin, has been kept at a high frequency because of its protective effect against UTIs.

Autosomal dominant tubulointerstitial kidney disease: diagnosis, classification, and management--A KDIGO consensus report.

Rare autosomal dominant tubulointerstitial kidney disease is caused by mutations in the genes encoding uromodulin (UMOD), hepatocyte nuclear factor-1ß (HNF1B), renin (REN), and mucin-1 (MUC1). Multiple names have been proposed for these disorders, including 'Medullary Cystic Kidney Disease (MCKD) type 2', 'Familial Juvenile Hyperuricemic Nephropathy (FJHN)', or 'Uromodulin-Associated Kidney Disease (UAKD)' for UMOD-related diseases and 'MCKD type 1' for the disease caused by MUC1 mutations. The multiplicity of these terms, and the fact that cysts are not pathognomonic, creates confusion. Kidney Disease: Improving Global Outcomes (KDIGO) proposes adoption of a new terminology for this group of diseases using the term 'Autosomal Dominant Tubulointerstitial Kidney Disease' (ADTKD) appended by a gene-based subclassification, and suggests diagnostic criteria. Implementation of these recommendations is anticipated to facilitate recognition and characterization of these monogenic diseases. A better understanding of these rare disorders may be relevant for the tubulointerstitial fibrosis component in many forms of chronic kidney disease.

Common variants in UMOD associate with urinary uromodulin levels: a meta-analysis.

Uromodulin is expressed exclusively in the thick ascending limb and is the most abundant protein excreted in normal urine. Variants in UMOD, which encodes uromodulin, are associated with renal function, and urinary uromodulin levels may be a biomarker for kidney disease. However, the genetic factors regulating uromodulin excretion are unknown. We conducted a meta-analysis of urinary uromodulin levels to identify associated common genetic variants in the general population. We included 10,884 individuals of European descent from three genetic isolates and three urban cohorts. Each study measured uromodulin indexed to creatinine and conducted linear regression analysis of approximately 2.5 million single nucleotide polymorphisms using an additive model. We also tested whether variants in genes expressed in the thick ascending limb associate with uromodulin levels. rs12917707, located near UMOD and previously associated with renal function and CKD, had the strongest association with urinary uromodulin levels (P<0.001). In all cohorts, carriers of a G allele of this variant had higher uromodulin levels than noncarriers did (geometric means 10.24, 14.05, and 17.67 µg/g creatinine for zero, one, or two copies of the G allele). rs12446492 in the adjacent gene PDILT (protein disulfide isomerase-like, testis expressed) also reached genome-wide significance (P<0.001). Regarding genes expressed in the thick ascending limb, variants in KCNJ1, SORL1, and CAB39 associated with urinary uromodulin levels. These data indicate that common variants in the UMOD promoter region may influence urinary uromodulin levels. They also provide insights into uromodulin biology and the association of UMOD variants with renal function.

A primary culture system of mouse thick ascending limb cells with preserved function and uromodulin processing.

The epithelial cells lining the thick ascending limb (TAL) of the loop of Henle perform essential transport processes and secrete uromodulin, the most abundant protein in normal urine. The lack of differentiated cell culture systems has hampered studies of TAL functions. Here, we report a method to generate differentiated primary cultures of TAL cells, developed from microdissected tubules obtained in mouse kidneys. The TAL tubules cultured on permeable filters formed polarized confluent monolayers in ∼12 days. The TAL cells remain differentiated and express functional markers such as uromodulin, NKCC2, and ROMK at the apical membrane. Electrophysiological measurements on primary TAL monolayers showed a lumen-positive transepithelial potential (+9.4 ± 0.8 mV/cm(2)) and transepithelial resistance similar to that recorded in vivo. The transepithelial potential is abolished by apical bumetanide and in primary cultures obtained from ROMK knockout mice. The processing, maturation and apical secretion of uromodulin by primary TAL cells is identical to that observed in vivo. The primary TAL cells respond appropriately to hypoxia, hypertonicity, and stimulation by desmopressin, and they can be transfected. The establishment of this primary culture system will allow the investigation of TAL cells obtained from genetically modified mouse models, providing a critical tool for understanding the role of that segment in health and disease.

Protein trafficking defects in inherited kidney diseases.

The nephron, the basic structural and functional unit of the kidney, is lined by different, highly differentiated polarized epithelial cells. Their concerted action modifies the composition of the glomerular ultrafiltrate through reabsorption and secretion of essential solutes to finally produce urine. The highly specialized properties of the different epithelial cell types of the nephron are remarkable and rely on the regulated delivery of specific proteins to their final subcellular localization. Hence, mutations affecting sorting of individual proteins or inactivating the epithelial trafficking machinery have severe functional consequences causing disease. The presence of mutations leading to protein trafficking defect is indeed a mechanism of pathogenesis seen in an increasing number of disorders, including about one-third of monogenic diseases affecting the kidney. In this review, we focus on representative diseases to discuss different molecular mechanisms that primarily lead to defective protein transport, such as endoplasmic reticulum retention, mistargeting, defective endocytosis or degradation, eventually resulting in epithelial cell and kidney dysfunction. For each disease, we discuss the type of reported mutations, their molecular and cellular consequences and possible strategies for therapeutic intervention. Particular emphasis is given to new and prospective therapies aimed at rescuing the trafficking defect at the basis of these conformational diseases.

Haploinsufficiency of ATP1A2 encoding the Na+/K+ pump alpha2 subunit associated with familial hemiplegic migraine type 2.

Headache attacks and autonomic dysfunctions characterize migraine, a very common, disabling disorder with a prevalence of 12% in the general population of Western countries. About 20% of individuals affected with migraine experience aura, a visual or sensory-motor neurological dysfunction that usually precedes or accompanies the headache. Although the mode of transmission is controversial, population-based and twin studies have implicated genetic factors, especially in migraine with aura. Familial hemiplegic migraine is a hereditary form of migraine characterized by aura and some hemiparesis. Here we show that mutations in the gene ATP1A2 that encodes the alpha2 subunit of the Na+/K+ pump are associated with familial hemiplegic migraine type 2 (FHM2) linked to chromosome 1q23 (OMIM 602481). Functional data indicate that the putative pathogenetic mechanism is triggered by a loss of function of a single allele of ATP1A2. This is the first report associating mutations of Na+K+ pump subunits to genetic diseases.

Leader peptide or pro-segment mutants of renin are misrouted to mitochondria in autosomal dominant tubulointerstitial kidney disease.

Autosomal dominant tubulointerstitial kidney disease (ADTKD), a rare genetic disorder characterised by progressive chronic kidney disease, is caused by mutations in different genes, including REN, encoding renin. Renin is a secreted protease composed of three domains: the leader peptide that allows insertion in the endoplasmic reticulum (ER), a pro-segment regulating its activity, and the mature part of the protein. Mutations in mature renin lead to ER retention of the mutant protein and to late-onset disease, whereas mutations in the leader peptide, associated with defective ER translocation, and mutations in the pro-segment, leading to accumulation in the ER-to-Golgi compartment, lead to a more severe, early-onset disease. In this study, we demonstrate a common, unprecedented effect of mutations in the leader peptide and pro-segment as they lead to full or partial mistargeting of the mutated proteins to mitochondria. The mutated pre-pro-sequence of renin is necessary and sufficient to drive mitochondrial rerouting, mitochondrial import defect and fragmentation. Mitochondrial localisation and fragmentation were also observed for wild-type renin when ER translocation was affected. These results expand the spectrum of cellular phenotypes associated with ADTKD-associated REN mutations, providing new insight into the molecular pathogenesis of the disease.

Allelic effects on uromodulin aggregates drive autosomal dominant tubulointerstitial kidney disease.

Missense mutations in the uromodulin (UMOD) gene cause autosomal dominant tubulointerstitial kidney disease (ADTKD), one of the most common monogenic kidney diseases. The unknown impact of the allelic and gene dosage effects and fate of mutant uromodulin leaves open the gap between postulated gain-of-function mutations, end-organ damage and disease progression in ADTKD. Based on two prevalent missense UMOD mutations with divergent disease progression, we generated UmodC171Y and UmodR186S knock-in mice that showed strong allelic and gene dosage effects on uromodulin aggregates and activation of ER stress and unfolded protein and immune responses, leading to variable kidney damage. Deletion of the wild-type Umod allele in heterozygous UmodR186S mice increased the formation of uromodulin aggregates and ER stress. Studies in kidney tubular cells confirmed differences in uromodulin aggregates, with activation of mutation-specific quality control and clearance mechanisms. Enhancement of autophagy by starvation and mTORC1 inhibition decreased uromodulin aggregates. These studies substantiate the role of toxic aggregates as driving progression of ADTKD-UMOD, relevant for therapeutic strategies to improve clearance of mutant uromodulin.

Activation of the bumetanide-sensitive Na+,K+,2Cl- cotransporter (NKCC2) is facilitated by Tamm-Horsfall protein in a chloride-sensitive manner.

Active transport of NaCl across thick ascending limb (TAL) epithelium is accomplished by Na(+),K(+),2Cl(-) cotransporter (NKCC2). The activity of NKCC2 is determined by vasopressin (AVP) or intracellular chloride concentration and includes its amino-terminal phosphorylation. Co-expressed Tamm-Horsfall protein (THP) has been proposed to interact with NKCC2. We hypothesized that THP modulates NKCC2 activity in TAL. THP-deficient mice (THP(-/-)) showed an increased abundance of intracellular NKCC2 located in subapical vesicles (+47% compared with wild type (WT) mice), whereas base-line phosphorylation of NKCC2 was significantly decreased (-49% compared with WT mice), suggesting reduced activity of the transporter in the absence of THP. Cultured TAL cells with low endogenous THP levels and low base-line phosphorylation of NKCC2 displayed sharp increases in NKCC2 phosphorylation (+38%) along with a significant change of intracellular chloride concentration upon transfection with THP. In NKCC2-expressing frog oocytes, co-injection with THP cRNA significantly enhanced the activation of NKCC2 under low chloride hypotonic stress (+112% versus +235%). Short term (30 min) stimulation of the vasopressin V2 receptor pathway by V2 receptor agonist (deamino-cis-D-Arg vasopressin) resulted in enhanced NKCC2 phosphorylation in WT mice and cultured TAL cells transfected with THP, whereas in the absence of THP, NKCC2 phosphorylation upon deamino-cis-D-Arg vasopressin was blunted in both systems. Attenuated effects of furosemide along with functional and structural adaptation of the distal convoluted tubule in THP(-/-) mice supported the notion that NaCl reabsorption was impaired in TAL lacking THP. In summary, these results are compatible with a permissive role for THP in the modulation of NKCC2-dependent TAL salt reabsorptive function.

Urinary secretion and extracellular aggregation of mutant uromodulin isoforms.

Uromodulin is exclusively expressed in the thick ascending limb and is the most abundant protein secreted in urine where it is found in high-molecular-weight polymers. Its biological functions are still elusive, but it is thought to play a protective role against urinary tract infection, calcium oxalate crystal formation, and regulation of water and salt balance in the thick ascending limb. Mutations in uromodulin are responsible for autosomal-dominant kidney diseases characterized by defective urine concentrating ability, hyperuricemia, gout, tubulointerstitial fibrosis, renal cysts, and chronic kidney disease. Previous in vitro studies found retention in the endoplasmic reticulum as a common feature of all uromodulin mutant isoforms. Both in vitro and in vivo we found that mutant isoforms partially escaped retention in the endoplasmic reticulum and reached the plasma membrane where they formed large extracellular aggregates that have a dominant-negative effect on coexpressed wild-type protein. Notably, mutant uromodulin excretion was detected in patients carrying uromodulin mutations. Thus, our results suggest that mutant uromodulin exerts a gain-of-function effect that can be exerted by both intra- and extracellular forms of the protein.

A transgenic mouse model for uromodulin-associated kidney diseases shows specific tubulo-interstitial damage, urinary concentrating defect and renal failure.

Uromodulin-associated kidney diseases (UAKD) are autosomal-dominant disorders characterized by alteration of urinary concentrating ability, tubulo-interstitial fibrosis, hyperuricaemia and renal cysts at the cortico-medullary junction. UAKD are caused by mutations in UMOD, the gene encoding uromodulin. Although uromodulin is the most abundant protein secreted in urine, its physiological role remains elusive. Several in vitro studies demonstrated that mutations in uromodulin lead to endoplasmic reticulum (ER) retention of mutant protein, but their relevance in vivo has not been studied. We here report on the generation and characterization of the first transgenic mouse model for UAKD. Transgenic mice that express the C147W mutant uromodulin (Tg(Umod)(C147W)), corresponding to the well-established patient mutation C148W, were compared with expression-matched transgenic mice expressing the wild-type protein (Tg(Umod)(wt)). Tg(Umod)(C147W) mice recapitulate most of the UAKD features, with urinary concentrating defect of renal origin and progressive renal injury, i.e. tubulo-interstitial fibrosis with inflammatory cell infiltration, tubule dilation and specific damage of the thick ascending limb of Henle's loop, leading to mild renal failure. As observed in patients, Tg(Umod)(C147W) mice show a marked reduction of urinary uromodulin excretion. Mutant uromodulin trafficking to the plasma membrane is indeed impaired as it is retained in the ER of expressing cells leading to ER hyperplasia. The Tg(Umod)(C147W) mice represent a unique model that recapitulates most of the features associated with UAKD. Our data clearly demonstrate a gain-of-toxic function of uromodulin mutations providing insights into the pathogenetic mechanism of the disease. These findings may also be relevant for other tubulo-interstitial or ER-storage disorders.

Uromodulin is expressed in renal primary cilia and UMOD mutations result in decreased ciliary uromodulin expression.

Uromodulin (UMOD) mutations are responsible for three autosomal dominant tubulo-interstitial nephropathies including medullary cystic kidney disease type 2 (MCKD2), familial juvenile hyperuricemic nephropathy and glomerulocystic kidney disease. Symptoms include renal salt wasting, hyperuricemia, gout, hypertension and end-stage renal disease. MCKD is part of the 'nephronophthisis-MCKD complex', a group of cystic kidney diseases. Both disorders have an indistinguishable histology and renal cysts are observed in either. For most genes mutated in cystic kidney disease, their proteins are expressed in the primary cilia/basal body complex. We identified seven novel UMOD mutations and were interested if UMOD protein was expressed in the primary renal cilia of human renal biopsies and if mutant UMOD would show a different expression pattern compared with that seen in control individuals. We demonstrate that UMOD is expressed in the primary cilia of renal tubules, using immunofluorescent studies in human kidney biopsy samples. The number of UMOD-positive primary cilia in UMOD patients is significantly decreased when compared with control samples. Additional immunofluorescence studies confirm ciliary expression of UMOD in cell culture. Ciliary expression of UMOD is also confirmed by electron microscopy. UMOD localization at the mitotic spindle poles and colocalization with other ciliary proteins such as nephrocystin-1 and kinesin family member 3A is demonstrated. Our data add UMOD to the group of proteins expressed in primary cilia, where mutations of the gene lead to cystic kidney disease.