Advances in uromodulin biology and potential clinical applications

Abstract

Uromodulin (also known as Tamm-Horsfall protein) is a kidney-specific glycoprotein secreted bidirectionally into the urine and circulation, and is the most abundant protein in normal urine. Although the discovery of uromodulin predates modern medicine, its significance in health and disease has been rather enigmatic. Evolving studies have gradually revealed that uromodulin exists in multiple forms and has important roles in urinary and systemic homeostasis. Most uromodulin in urine is polymerized into highly organized filaments, whereas non-polymerizing uromodulin is detected in both urine and circulation, and can have distinct roles. The interactions of uromodulin with the immune system, which were initially reported to be a key role of this protein, are now better understood. Moreover, the discovery that uromodulin is associated with a spectrum of kidney diseases, including acute kidney injury, chronic kidney disease and autosomal dominant tubulointerstitial kidney disease, has further accelerated investigations on the role of uromodulin. These discoveries have prompted new questions and ushered in a novel era in uromodulin research. Here, we delineate the latest discoveries in uromodulin biology, its emerging roles in modulating kidney and systemic diseases, and consider future directions, including its potential clinical applications.

Introduction

Uromodulin (also known as Tamm-Horsfall protein) is a kidney-specific protein encoded by UMOD and is the most abundant urinary protein in physiological states.^1–3 Uromodulin is also a well-known major component of urinary casts.⁴ The history of uromodulin is extensive, tracing back to the 19th century. In 1873, Rovida described cilindrina as a major constituent of hyaline casts.⁵ In 1950, Tamm and Horsfall isolated a urinary protein that inhibited viral hemagglutination and named it Tamm-Horsfall protein.⁶ In 1985, Muchmore and Decker identified a urinary immunomodulatory protein from the urine and named it uromodulin.⁷ Subsequently, Pennica and colleagues showed that uromodulin and Tamm-Horsfall protein are the same protein.⁸ Over decades, extensive research has gradually revealed the pleiotropic roles of uromodulin in physiology and pathology. In particular, uromodulin research was accelerated by the discovery in the early 2000s that common UMOD polymorphisms are associated with chronic kidney disease (CKD) and that rare UMOD mutations can cause autosomal dominant tubulointerstitial kidney disease (ADTKD). The latest innovations in uromodulin biology include a better understanding of its expression and secretion mechanisms, the recognition of its multiple forms, enhanced of understanding of structural data, advances in examining its potential as a functional biomarker, recognition of a systemic role for this protein beyond the kidney and a role in acute kidney injury (AKI), as well as elucidation of the pathophysiology ADTKD and evolving therapeutic strategies for this disease.

In this Review, we summarize the latest innovations in uromodulin biology. First, we consider uromodulin physiology, including its regulation and function. An emerging concept is that uromodulin, which was traditionally recognized as a urinary secretory protein, can be secreted in different polymeric and non-polymeric forms and has multiple protective roles in the interstitium, circulation and intracellularly. We then describe the crosstalk between uromodulin and the immune system, which was originally reported as a major role for this protein. Emerging evidence of uromodulin as an attractive clinical biomarker will also be summarized. Finally, we describe advances in understanding the role of uromodulin in the pathogenesis of kidney diseases, focusing on AKI, CKD, ADTKD and hypertension, and their clinical implications.

Advances in uromodulin biology

Expression and localization

One of the main characteristics of uromodulin is its highly localized and abundant expression in the kidney, which suggests a key role in this organ.⁹ Uromodulin is expressed predominantly in the cells of the thick ascending limb (TAL) of the loop of Henle and, to a lesser extent, in the cells of early distal convoluted tubules (DCT) (Figure 1A).^10,11 Uromodulin is one of the most abundant transcripts in rat and mouse TAL cells^12,13 and in bulk human kidney samples.¹⁴ Within cells, uromodulin is predominantly localized at the apical membrane, with a small portion found on the basolateral membrane (Figure 1B, left).^10,15

Figure 1: Expression, function and structure of uromodulin.

(A) Uromodulin is expressed in the cells of thick ascending limbs (TAL) of the loop of Henle and early distal convoluted tubules (DCT), which are highlighted in red. PT = proximal tubule; LH = loop of Henle; mTAL = medullary TAL; cTAL = cortical TAL; CD = collecting ducts. (B) Uromodulin is mainly localized at the apical membrane and is secreted into the urine. A small portion of uromodulin is targeted to the basolateral membrane and is released into the interstitium, PT cells and circulation. Uromodulin is a multi-functional protein, and its function depends on its site of action and form. UTIs = urinary tract infections. NKCC2 = sodium potassium chloride cotransporter 2; ROMK = renal outer medullary potassium channel; NCC = sodium chloride cotransporter. (C) Primary structure and domains of uromodulin. The Roman numerals 1 through 4 indicate the four EGF-like domains; D10C = domain with conserved ten cysteines; ZP = zona pellucida; IHP = internal hydrophobic patch; EHP = external hydrophobic patch; GPI = glycosylphosphatidylinositol.

UMOD is located on chromosome 16p12.3–16p13.11.¹⁶ and its promoter is predicted to be regulated by a large network of interacting transcription factors.¹⁷ Hepatic nuclear factor 1β (HNF1B) positively regulates Umod transcription in mice.¹⁸ Single-nucleotide polymorphisms (SNPs) spanning the promoter region of UMOD are also associated with uromodulin expression. The representative SNPs (rs4293393 and rs12917707) are in complete linkage disequilibrium, and the major allele (T at rs4293393 and C at rs12917707 on the reverse strand) is linked to high UMOD mRNA expression in the kidney¹⁹ and high urinary uromodulin secretion.^19–22 The effect of the rs4293393 SNP on UMOD gene expression was confirmed in vitro using luciferase reporter assay.¹⁹ The UMOD promoter region includes predicted glucocorticoid response elements and promoter activity was responsive to glucocorticoid exposure.¹⁹ The rs77924615 SNP in the flanking gene PDILT is also associated with UMOD but not with PDILT expression.^23–25 We further reported the presence of predicted oestrogen response elements in the human and mouse UMOD loci, and Umod expression was oestrogen-responsive in immortalized mouse kidney TAL (MKTAL) cells,²⁶ which is consistent with sexual differences in uromodulin levels (discussed below). Of note, dietary salt enhances Umod transcription in rats²⁷, and salt intake and urinary uromodulin levels are positively associated in mice²⁸ and humans^29–31. MicroRNAs (miRNAs) can also modulate Umod expression — rat pre-miR-16 and pre-miR-195 decreased luciferase activity of reporter vectors harboring the Umod 3’- untranslated region (UTR) sequence in HeLa cells.³²

In addition to TAL and early DCT cells, uromodulin has been detected in human glomeruli using immunohistochemical and fluorescence methods^9,33,34. One commonly proposed explanation is that uromodulin flows back across the loop of Henle and the entire proximal segment, and attaches to Bowman’s capsule.³⁴ Glomerular uromodulin is also detectable in unilateral ureteral obstruction (UUO) mouse models^35,36, potentially supporting the backflow hypothesis. However, Umod mRNA was also detected in microdissected glomeruli of rats¹², which raises the possibility of de novo synthesis of uromodulin in glomeruli. This potential ‘ectopic’ expression must be validated with other techniques (for example, in situ hybridization or spatial transcriptomics). Further investigation is also needed to evaluate whether glomerular uromodulin is biologically relevant, whether glomerular cells can synthesize uromodulin and whether circulating uromodulin can cross the glomerular filtration barrier in disease and becomes involved in this observation.

Beyond the kidney, uromodulin is reportedly expressed in intestinal microfold cells (M cells), which are embedded in the intestinal epithelium and are responsible for phagocytosis and transcytosis of antigens and pathogens across epithelium.³⁷ One study showed that uromodulin in M cells functions as an uptake receptor for a probiotic strain Lactobacillus acidophilus L-92.³⁸ Uromodulin might be involved in gut microbiome homeostasis and gut dysbiosis is known to contribute to kidney diseases including CKD, hypertension and kidney stones.^39–41 Given that these diseases are also associated with kidney uromodulin expression (discussed below), studying disease-associated alterations in intestinal uromodulin expression and their potential impact on kidney disease might offer a novel angle into its pathogenesis and inform therapeutic strategies. Uromodulin might also be expressed ectopically in other organs and cell types under certain pathological conditions such as inflammation, infection or stress. For example, uromodulin protein has been detected in bronchoalveolar lavage (BAL) fluid from patients with acute respiratory distress syndrome (ARDS)⁴² or mice with talc-induced granulomas.⁴³ Whether these findings reflect de novo synthesis or concentration of circulating uromodulin remains unclear.

Primary structure and protein domains

Uromodulin is a glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein that is secreted into urine and circulation. The uromodulin precursor protein comprises 640 amino acids (FIG.1c), most of which are located extracellularly, and the C-terminal side attaches to the cell membrane via GPI anchoring.⁴⁴ At the N terminus, uromodulin contains a signal peptide (residues 1–24) that guides this protein into the endoplasmic reticulum (ER) membrane and, subsequently, the secretory pathway. Four epidermal growth factor (EGF)-like domains are believed to be important for protein–protein interactions,¹ but their exact role remains elusive. A domain with ten conserved cysteines (D10C) was recently redefined⁴⁵ in 2022, after being initially reported to have eight conserved cysteines (D8C).⁴⁶ Other domains include a bipartite Zona Pellucida (ZP) domain, which comprises ZP-N and ZP-C sub-domains and is crucial for extracellular polymerization. Finally, the internal hydrophobic patch (IHP) and external hydrophobic patch (EHP) motifs⁴⁷ interact to prevent intracellular polymerization (also referred to as aggregation) of uromodulin during intracellular trafficking; these motifs also modulate the extracellular secretion of polymerizing uromodulin.^47,48

Intracellular processing and trafficking

Within the secretory pathway, uromodulin undergoes GPI anchoring, formation of 24 disulfide bonds and glycosylation. Uromodulin is a highly glycosylated protein, with added glycans comprising 30% of its total molecular weight. Initially, eight N-linked glycosylation sites (N38, N76, N80, N232, N275, N322, N396 and N513) were predicted and all except N38 were found to be glycosylated.⁴⁹ However, another study reported that N38 is also glycosylated.⁵⁰ These studies used isolated human urinary uromodulin from donors and their discrepant findings might be explained by the interindividual variability in glycosylation. Differences in the ratio of urinary polymeric-to-non-polymeric uromodulin (discussed below) during purification might also contribute to conflicting results. Most uromodulin glycans are of the complex type, but N275 contains a high-mannose glycan (part of N513 is also reported to be modified with high-mannose glycans).⁵⁰ The high-mannose glycans at N275 are crucial for protection against urinary tract infections (discussed below). Uromodulin also undergoes O-linked glycosylation,⁵¹ but the significance and sites of O-linked glycosylation have not been established. GPI anchors, N-glycans and O-glycans are all known apical sorting signals in polarized epithelial cells,⁵² but which of these mechanisms is most crucial for apical trafficking of uromodulin remains unclear.

Calcium-sensing receptor (CaSR) activity negatively regulates the apical trafficking of uromodulin in mice and in primarily isolated mouse TAL cells. Mechanistically, intracellular cyclic AMP (cAMP) signaling, which is downregulated by CaSR, induced uromodulin trafficking.⁵³ In addition, knockdown of KRT40, which encodes cytoskeletal protein keratin-40, inhibits apical trafficking of uromodulin in primary isolated mouse TAL cells.⁵⁴ This finding is supported by the genome-wide association study (GWAS) finding that SNPs in the KRT40 locus are associated with urinary secretion of uromodulin.⁵⁴ In mice, conditional inactivation of Myh9 and Myh10, which encode cytoskeletal protein myosin 9 and 10, respectively, also inhibited the apical trafficking of uromodulin in mice. The localization of sodium potassium chloride co-transporter 2 (NKCC2), renal outer medullary potassium channel (ROMK), and Na⁺,K⁺-ATPase in TAL cells remained normal, indicating a specific role for myosins 9 and 10 on uromodulin trafficking.⁵⁵ A subsequent report showed that inhibition of microtubules hampered the trafficking of uromodulin in vitro.⁵⁶ These results suggest an interplay between the cytoskeleton and uromodulin, but whether this interaction is direct or indirect requires further investigation.

No evidence suggests that the intracellular protein degradation system regulates uromodulin expression except in the case ADTKD-causing mutant uromodulin (discussed later). A 2023 study showed that wild-type intracellular uromodulin abundance was unchanged after inhibition of autophagy or proteasome activity in murine inner medullary collecting duct (mIMCD) cells that overexpressed exogenous uromodulin.⁵⁷ This finding suggests that, under physiological conditions, uromodulin expression is mostly regulated by its production and extracellular secretion, rather than intracellular degradation.

Polymerization and extracellular secretion

Another key feature of uromodulin is that it polymerizes into highly organized filaments when released from the apical membrane (Figure 2A, B). In a 2020 report⁵⁰ cryo-electron tomography (cryoET) data showed that uromodulin filaments create a ‘fishbone’-like structure composed of a zig-zag-shaped core and protruding arms. The core is formed by polymerized ZP domains, and the arms are composed of EGF-like I–III and D8C (now known as D10C) domains; EGF-like IV domains connect the arms to the core.⁵⁰ The polymerization process involves the release of apical membrane uromodulin into the urine by the serine protease hepsin (Figure 2A).⁵⁸ Removal of the EHP motif by this cleavage induces a conformational change in the ZP-N–ZP-C linker region and homodimerization of ZP domains.⁵⁹ Subsequent studies used cryo-electron microscopy (cryoEM) and to investigate the polymerization mechanism of uromodulin.^48,60 Although there are differences regarding the order of hepsin cleavage and homodimerization of membrane-bound uromodulin in the proposed models in the two cryo EM studies, these results suggest that the dynamic conformational change of ZP domains and hepsin cleavage occur synchronously in membrane-bound uromodulin.

Figure 2: Distinct pathways of extracellular secretion of uromodulin.

(A) The mechanism of extracellular secretion of uromodulin. Most uromodulin is secreted into the urine and undergoes polymerization (upper left). Uromodulin is a glycosylphosphatidylinositol (GPI)-anchored protein that is mainly sorted to the apical membrane. At the apical membrane, hepsin plays a key role in the secretion of urinary polymerizing uromodulin. Hepsin cleaves the C terminus of uromodulin, including the external hydrophobic patch (EHP) motif. EHP is a polymerization-blocking motif and its removal enables polymerization of uromodulin into filaments. Several molecules and stimuli regulate this secretion. Non-polymerizing uromodulin is secreted into the urine and circulation via alternative pathways (upper right and bottom). Non-polymerizing uromodulin retains the EHP motif and therefore does not polymerize in the urine and circulation. The mechanisms regulating the secretion of non-polymerizing uromodulin secretion are largely unknown..(B) Immunofluorescence of uromodulin filaments above the apical surface of Madin-Darby canine kidney (MDCK) cells stably expressing human uromodulin. Scale bar = 10 μm. (C) SDS-PAGE of human urinary uromodulin shows a single broad band. (D) Native-PAGE of human urinary uromodulin separates polymerizing and non-polymerizing uromodulin. (E) The C-terminal amino acid sequence of human uromodulin. Polymerizing uromodulin is cleaved prior to the EHP motif by hepsin, which enables its polymerization into filaments. SNP, single-nucleotide polymorphism.

Experimental evidence indicates that several regulators of urinary uromodulin secretion exist (Figure 2A). Short-term vasopressin treatment induced urinary secretion of polymerizing uromodulin and reduced membrane uromodulin abundance in Madin-Darby canine kidney (MDCK) cells and mice.⁶¹ This finding is consistent with previous data showing lower abundance of membrane-bound uromodulin after a 5-day desmopressin treatment in rats.⁶² However, chronic treatment slightly decreased urinary uromodulin secretion in rats,⁶³ indicating a time-course-dependent effect of vasopressin. We also showed that water loading increases urinary secretion of polymerizing uromodulin³⁰, which suggests that urinary flow, hypotonic stimuli, as well as vasopressin, could be involved in uromodulin secretion. Finally, inhibition of ROMK decreased the apical secretion of uromodulin and increased intracellular uromodulin abundance in TAL cells and in mice.⁶⁴ This role is supported by the GWAS finding that SNP rs28555800 in the KCNJ1 gene (encoding ROMK) is associated with urinary secretion of uromodulin.²²

Extracellular secretion of non-polymerizing uromodulin

The kidney also releases non-polymerizing uromodulin into urine and circulation. We reported that non-polymerizing uromodulin is cleaved at an alternative site, distal to the EHP motif. Therefore, non-polymerizing uromodulin retains the EHP motif, which maintains the protein in a polymerization-incompetent state (Figure 2A).⁶⁵ Urinary uromodulin forms a single band in SDS-PAGE (Figure 2C) but is separated into two forms in native-PAGE (Figure 2D). In SDS-PAGE, the molecular weight of non-polymerizing uromodulin is higher (~100 kDa) than that of polymerizing uromodulin (~85 kDa) owing to its distal C-terminal cleavage. However, the two forms cannot be distinguished by SDS-PAGE given that the abundant protein glycosylation creates a broad band. Polymeric uromodulin usually does not enter the gels in native-PAGE because its molecular weight exceeds a million Daltons. Of note, polymerizing uromodulin sometimes enters the gels and is detected as a laddering of high-molecular-weight bands, likely due to partial dissociation or degradation related to storage. The molecular weight of native urinary non-polymerizing uromodulin is ~200 kDa (Figure 2D), suggesting dimerization, although its detailed structure is unknown. SDS-PAGE combined with N-deglycosylation^{28,47,58,64,66} or with ultracentrifugation^28,61,66,67 can also separate these two forms.

Secretion of non-polymerizing uromodulin is independent of hepsin given that it is still released in mice lacking hepsin.^28,58 The underlying biological mechanism governing the alternative cleavage of urinary non-polymerizing uromodulin is under investigation. We showed that the C terminus of urinary non-polymerizing uromodulin is F617, which is beyond the GPI anchoring site (Figure 2E)⁶⁵, suggesting that urinary non-polymerizing uromodulin is not a GPI anchored protein. In urine, non-polymeric uromodulin is usually a minor form, but its abundance increases relatively in concentrated urine owing to decreased polymerizing uromodulin.³⁰ This ratio change might help to prevent urinary cast formation in the setting of urinary concentration (for example, owing to dehydration). Urinary non-polymeric uromodulin, which was evaluated by SDS-PAGE after N-deglycosylation, is also more abundant in rats with pregnancy-induced hypertension than in pregnant control strain rats, although the molecular mechanism driving this change is unknown.⁶⁶ Of note, peptides comprising the hepsin cleavage site and the GPI anchoring site have been identified in urine through mass spectrometry.^3,66,68 However, whether these peptides indicate degradation of non-polymerizing uromodulin, the shedding of C-terminal cell surface uromodulin following hepsin cleavage or an additional distinct form of uromodulin remains unclear.

A small amount of uromodulin is targeted to the basolateral membrane and secreted into the interstitium and circulation.^2,15,69 We showed that non-polymerizing uromodulin is the dominant form present in the circulation using size exclusion chromatography.⁷⁰ Of note, highly polymerizing (that is, aggregated) proteins are usually immunogenic in circulation. Circulating uromodulin retains the EHP motif and its C-terminus is at R606 or K607, which precede the GPI-anchoring site (Figure 2E)⁶⁵, thereby differing from both polymerizing and non-polymerizing uromodulin in the urine. The regulatory mechanisms of circulating uromodulin are different from those of urinary uromodulin, at least in certain conditions.^30,61 These findings suggest that circulating uromodulin is not the result of leakage of non-polymeric urinary uromodulin, but is rather produced by an alternative pathway. The biological mechanism governing basolateral targeting or secretion of circulating uromodulin is still largely unknown, and putative basolateral sorting signals in the uromodulin sequence have not yet been characterized. We found that basolateral translocation and secretion of uromodulin are enhanced during recovery from kidney ischaemia–reperfusion injury (IRI)⁶⁹ and sepsis⁴². Importantly, the localization of Na⁺,K⁺-ATPase and NKCC2 was preserved in TAL cells after IRI, excluding the possibility that increased basolateral targeting results from the loss of epithelial polarity. This finding suggests that stress or repair processes might induce basolateral targeting and secretion of uromodulin, although the underlying mechanisms remain unknown. A GWAS meta-analysis of circulating uromodulin revealed that a missense variant of B4GALNT2, which encodes a glycosylating enzyme, is associated with higher circulating uromodulin levels, suggesting a role for differential glycosylation in basolateral targeting and secretion.²³

Versatile functions of uromodulin

Analysis of Umod-knockout mice has revealed its pleiotropic functions, most of which are supported by clinical observations in humans. Below we consider how these versatile roles are influenced by the form and sites of action of uromodulin (Figure 1B, right).

Urinary uromodulin

Urinary uromodulin has multiple protective roles in the tubular lumen. Umod-knockout mice have increased susceptibility to urinary tract infections (UTIs) caused by type 1-fimbriated E. coli^71,72 and other bacteria.^73–75 Uromodulin filaments form a ‘fishing net’-like structure that can trap bacteria.⁷⁶ For example, uromodulin filaments bind to type 1-fimbriated E. coli and compete for its binding and invasion into the urothelium.⁷⁷ CryoET experiments demonstrated that high-mannose glycans at uromodulin N275 bind to FimH, which is a mannose-specific adhesin expressed by type 1-fimbriated E. coli.⁵⁰ Moreover, encapsulation by uromodulin filaments prevented the urothelial invasion by E. coli and was also effective in inducing bacterial aggregation and clearance.⁵⁰ Another study combined AlphaFold2 predictions, X-ray crystallography and cryoEM, and examined the interaction between uromodulin and E. coli at high resolution. Their findings showed that uromodulin harbors a decoy module comprised of a β-hairpin and the D10C domain. This module protects high-mannose glycans N275 from maturation to complex-type glycans and provides an interface between uromodulin and FimH.⁴⁵ These protective roles of uromodulin are supported by the clinical observations that higher urinary uromodulin concentration is linked to a lower risk of UTIs⁷⁸ and decreased levels of UTI markers.⁷⁹

Protection against kidney stones is another major role of uromodulin. Uromodulin inhibits crystal aggregation of calcium oxalate and calcium phosphate,^80–82 which are major components of urinary stones, and Umod-knockout mice develop calcium crystals and calcification in the kidney.^83–85 Uromodulin also promotes urinary calcium reabsorption by interacting with the calcium channel transient receptor potential cation channel subfamily V member 5 (TRPV5) in DCT cells.⁸⁶ These protective roles are supported by a GWAS study in which a UMOD SNP that drives higher uromodulin levels was associated with a lower risk of stone formation.⁸⁷ Urinary uromodulin also increases urinary magnesium reabsorption by interacting with the magnesium channel TRPM6 in DCT cells.⁸⁸

Whether urinary polymeric and non-polymeric uromodulin have overlapping or distinct functions in the urine remains unclear. The protection against UTIs is probably conferred by polymerizing uromodulin, considering the importance of filament formation. Other roles reported for urinary uromodulin in studies that have used urinary uromodulin purified by salt precipitation,^80,81,86,88 might also be mediated by polymeric uromodulin, given that this isolation method seems to selectively isolate polymeric uromodulin.⁶⁵ However, this assumption must be investigated further as other studies^89,90 have reported the presence of monomeric uromodulin with biological activity within solutions of urinary uromodulin isolated by salt precipitation. The role of urinary non-polymeric uromodulin remains elusive, partially because this form has only become better characterized in the past decade and the low concentration of this isoform relative to polymeric uromodulin complicates its isolation. We were able to isolate a monomeric truncated form of uromodulin that terminates at amino acid 434 using size exclusion chromatography.⁷⁰ The absence of polymerization is probably due to the lack of interplay between the bipartite ZP domains as this form is truncated at the end of ZP-N sub-domain. Of note, this truncation might result from urinary proteolysis and/or handling and might not be physiologically relevant in the urine. Although it lacks the ZP-C domain and one of the N-glycosylation sites (N513), this truncated uromodulin contains most uromodulin domains (EGF-like, D10C and ZP-N domains), and might be useful as a mimic of non-polymeric uromodulin.^42,70 An in vivo mouse model that selectively lacks non-polymeric uromodulin would also be useful but is unlikely to be developed without the identification of the molecules that regulate the secretion of urinary non-polymerizing uromodulin.

Circulating (non-polymeric) uromodulin

Emerging evidence suggests that uromodulin secreted basolaterally has important roles in neighboring interstitial cells and proximal tubular cells, as well as systemic effects in the circulation. In the interstitium and proximal tubular cells, uromodulin can regulate immune cells and inhibit inflammatory signaling (discussed below). Moreover, we discovered a systemic antioxidant role for circulating uromodulin using an unbiased multi-omics approach. Circulating uromodulin inhibits the cation channel TRPM2 and decreases reactive oxygen species (ROS) production in the kidneys, lungs and circulation.⁹¹ Another study showed that circulating uromodulin inhibits vascular calcification by interacting with and inhibiting pro-inflammatory cytokine signaling in the circulation.⁹² These protective roles of uromodulin are supported by clinical observations that decreased circulating uromodulin levels are associated with increased mortality and cardiovascular complications (discussed below).

Intracellular uromodulin

Within TAL and DCT cells, uromodulin has a key role in salt handling. Studies of Umod-knockout and overexpressing mice indicate that uromodulin positively regulates the activity of NKCC2^19,93 and ROMK⁹⁴ in TAL cells, and sodium–chloride cotransporter (NCC)¹¹ in DCT cells. Mechanistically, uromodulin facilitates the phosphorylation of NKCC2 and NCC by activating the SPAK (also known as STK39)– serine/threonine-protein kinase OSR1 signaling pathway^11,19. Uromodulin also downregulates tumour necrosis factor (TNF), which could contribute to NKCC2 activation.⁹⁵ The specific intracellular compartments where these interactions take place remain unclear. Of note, mutant uromodulin S614X, which lacks GPI-anchoring, failed to phosphorylate NKCC2¹⁹, suggesting a requirement for uromodulin association with the membrane within the secretory pathway.

Interactions of uromodulin with immune cells

Although uromodulin was originally discovered as an inhibitor of viral hemagglutination,⁶ it was rediscovered as a modulator of immune cell function in 1985.⁷ Early in vitro studies used urinary uromodulin, which might be predominantly polymeric, although these studies were not intended to distinguish the function of polymeric and non-polymeric uromodulin. Consequently, their findings about immune cell interactions cannot be easily extrapolated to immune cells in the kidney interstitium, circulation and other organs, where non-polymeric uromodulin is dominant. Here, we discuss the findings on the interaction between uromodulin and immune cells (Figure 3).

Figure 3: Interactions of uromodulin with immune cells.

The immunomodulatory role of uromodulin depends on the sites and form. Urinary polymerizing uromodulin down-regulates neutrophil activity to suppress excessive inflammation in the urinary tract. The interaction between uromodulin and an inhibitory receptor Siglec-9 on the neutrophil is important for this role. Uromodulin also acts on proximal tubule cells and down-regulates granulopoiesis by inhibiting the IL-23—IL-17 axis through neutrophils. Uromodulin deficiency increases IL-23 expression in proximal tubules, thereby acting on neutrophils and possibly other cells to producing IL-17, resulting in stimulating granulopoiesis by inducing granulocyte colony stimulating factor (G-CSF). Non-polymerizing uromodulin also promotes macrophage phagocytosis and proliferation in the interstitium and circulation. ROS = reactive oxygen species.

Interaction with neutrophils

Urinary uromodulin interacts with neutrophils⁹⁶ through Siglec-9, which is an inhibitory receptor expressed on neutrophils; this interaction suppressed neutrophil ROS production, chemotaxis and killing of bacteria in vitro.⁹⁷ These results suggest that uromodulin might protect the urinary tract from excessive inflammation by dampening neutrophil activity. Moreover, in an ischaemia-reperfusion injury model, Umod-knockout mice had enhanced kidney damage and expression of the neutrophil chemoattractant CXC-chemokine ligand 2 (CXCL2) in S3 proximal tubules, resulting in increased neutrophil infiltration.⁹⁸ Umod-knockout mice also have increased numbers of neutrophils in the kidney, liver and circulation, prior to injury; we identified activation of an IL-23–IL-17 axis and enhanced granulopoiesis as contributing factors.⁹⁹ Rupture of the tubular basement membrane in kidney diseases has been suggested to promote the deposition of polymeric uromodulin deposits in the extratubular interstitium^33,100,101 although these findings tend to be focally localized. Of note, the most conclusive evidence that polymeric uromodulin deposits in the interstitium comes from ultrastructural analysis of experimental obstructive nephropathy.³⁶ Overall, although the absence of foci of extravasated polymerizing uromodulin might contribute to the observations reported in Umod-knockout mice, the global changes observed within the renal parenchyma in uromodulin deficiency are attributed mainly to the non-polymerizing (interstitial or circulating) form.

Interaction with mononuclear phagocytes

Uromodulin can activate mononuclear phagocytes, including macrophages and dendritic cells. One report suggested that urinary uromodulin (purified by salt precipitation) activates dendritic cells by triggering Toll-like receptor 4 (TLR-4) signaling.¹⁰². Another study reported that uromodulin interacts with scavenger receptors on dendritic cells.¹⁰³ Urinary uromodulin can also trigger IL-1β secretion by monocytes and this effect is dependent on the NLRP3 inflammasome.¹⁰⁴ Another study reported that polymerized uromodulin- delivered by intrascrotal and intraperitoneal injection- acts as a damage-associated molecular pattern (DAMP), leading to local inflammation, leukocyte recruitment and TNF secretion by macrophages, as well as affecting vascular permeability and increasing neutrophil extravasation by¹⁰¹; the injection of uromodulin into a site where the protein is not usually expressed represents an important limitation. These pro-inflammatory functions ascribed to polymeric uromodulin contrast with our findings in Umod-knockout mice, which might reflect the activity of the non-polymeric form. Compared with wild-type controls, the kidneys of Umod-knockout mice had a lower number of mononuclear phagocytes and, in the setting of IRI, fewer of these cells had an anti-inflammatory (M2-like) phenotype.⁷⁰ Importantly, monomeric truncated uromodulinstimulated macrophage proliferation⁷⁰ and phagocytosis in vitro.⁴² Administration of monomeric truncated uromodulin to Umod-knockout mice with kidney IRI increased the number of mononuclear phagocytes in the kidney and mitigated the severity of injury.⁷⁰

Importance in sepsis

We found that the interaction between uromodulin and the immune system is protective in sepsis.⁴² Sepsis without severe kidney injury was associated with increased levels of circulating uromodulin in both patients and in an animal model, in which the rise in circulating uromodulin resulted from increased basolateral trafficking of the protein. Interestingly, uromodulin was also detected in the bronchoalveolar lavage (BAL) fluid of patients with acute respiratory distress syndrome (ARDS). In a cecal ligation and puncture model, Umod-knockout mice had increased mortality and bacterial burden compared with wild-type controls. Monomeric truncated uromodulin supplementation promoted phagocytosis and cell migration of macrophages, and improved survival.⁴²

Early evidence of immunomodulation

In the early 1980s, researchers at the Pasteur Institute in Paris isolated a protein from talc-induced granulomas in mice. When administered to mice, this protein could protect from a lethal infection with the bacteria Listeria monocytogenes.⁴³ Subsequently, antibodies raised against this protein were shown to react with a homologous protein in human urine.¹⁰⁵ A 92KDa protein extracted from this urinary protein (later confirmed to be uromodulin) also protected mice against lethal infection with L. monocytogenes, even in animals with severe combined immunodeficiency disease (SCID) lacking B and T lymphocytes, which suggested involvement of the innate immune system. The researcher also reported that this protective effect depended on the oligosaccharide moieties of the protein.⁸⁹ Although the original uromodulin isolated from urine by salt precipitation was also protective against Listeria, this effect was lost after purifying polymerizing uromodulin by ultracentrifugation, which the researchers suggested completely removed the 92KDa protein that conferred protection.⁹⁰ At the time, the researchers believed the 92-KDa protein to be distinct from uromodulin, but this extract might instead represent monomeric uromodulin⁶⁵. The presence of a considerable amount of non-polymeric uromodulin with biological activity within fractions of uromodulin originally purified by salt precipitation contrasts with findings that this purification method is selective for polymeric uromodulin⁶⁵; the reasons for this discrepancy remain unclear. If the protein isolated by Fontan and colleagues is non-polymeric uromodulin, their work would further suggest functional differences between polymeric and non-polymeric uromodulin on immune cells. Importantly, these studies also indicate a systemic protective role for non-polymeric uromodulin in infections.

Uromodulin as a biomarker

Biomarkers are measurable indicators of physiological or pathological states and can be used to assess the efficacy of a therapeutic intervention.¹⁰⁶ Emerging evidence indicates that uromodulin is an attractive biomarker of kidney function independent from traditional markers such as creatinine or cystatin C. Uromodulin is distinct because it is a kidney-specific protein that is exclusively produced by tubular cells, it can be easily measured from urine and serum with high reliability, sensitivity and specificity, and it is associated with a functional tubular response that partially depends on nephron mass.

Measurement of uromodulin levels

Circulating uromodulin is a stable antigen with a high concordance under various temperatures (−20°, 4° and 37°) even after five freeze and thaw cycles.¹⁰⁷ By contrast, uromodulin levels measured in human urine are greatly affected by centrifugation, vortexing and storage conditions.¹⁰⁸ Furthermore, polymerization of urinary uromodulin might partially mask the epitopes detected by uromodulin antibodies, thereby affecting quantification. Consequently, standard operating protocols are crucial for urinary uromodulin measurement.

Several methods can be used for measuring uromodulin levels (Table 1). Of note, most studies have not distinguished between urinary polymeric and non-polymeric uromodulin. Of note, unadjusted urinary uromodulin concentration is expected to be relevant to the functions of uromodulin in the urinary space, whereas urinary uromodulin concentration normalized to urine flow rate (measured as time) is indicative of uromodulin secretion. Urinary uromodulin concentration indexed to urinary creatinine is comparable to that normalized to flow rate, which is helpful when only spot urine samples are available rather than urine collected across specific time periods.^30,109

Table 1.

Methods for measuring the levels of uromodulin

Technique	High throughput	Distinction between polymeric and non-polymeric forms	Refs.
SDS-PAGE	No	No	108
SDS-PAGE after N-deglycosylation	No	Yes	28,47,58,64,66
SDS-PAGE after ultracentrifugation	No	Yes	28,61,66,67
Native-PAGE	No	Yes	30,65
Antibody-based ELISA	Yes	Yesa	108
Aptamer-based ELISA	Yes	No	23

^aWhen using antibodies against EHP-motif.⁶⁵ Abbreviation; ELISA = enzyme-linked immunosorbent assay; EHP = external hydrophobic patch.

Uromodulin levels and nephron mass

Given that uromodulin is exclusively produced by tubular cells, its abundance is thought to reflect nephron mass. Accordingly, uromodulin levels in urine^110–112 and circulation^{107,113–115} depend on nephron mass in cohorts of patients with kidney disease. However, most studies report that circulating uromodulin levels are not associated with glomerular filtration rate (GFR) in healthy individuals.^26,116 For example, one study of healthy kidney donors showed great variability in serum uromodulin levels between donors despite a relatively constant GFR.¹¹⁶ Other physiological or pathophysiological factors (for example, sex and metabolism), as well as genetic variation, probably regulate uromodulin expression, and the effect of kidney mass might only become apparent when kidney function declines.

Uromodulin levels and sexual dimorphism

Sexual dimorphism can influence levels of circulating uromodulin. Specifically, female sex is associated with higher serum uromodulin levels^{23,26,107,117}, although women have fewer nephrons than men.¹¹⁸ We reported that UMOD oestrogen-responsivity might explain this sexual dimorphism.²⁶ Sex differences in serum uromodulin levels found in adults but not in children¹⁰⁷ further support the hypothesis of oestrogen-mediated regulation. Further studies are needed to determine whether oestrogen only affects UMOD transcription or can also regulate its basolateral secretion. Considering its systemic roles, higher serum uromodulin in females might contribute to sexual dimorphism in health and disease, including differences in immune responses.¹¹⁹

Uromodulin levels and metabolic syndrome

Emerging evidence suggests a potential link between uromodulin levels and metabolic states. Lower circulating uromodulin levels might be associated with unfavorable glucose metabolism,^120,121 as well as the prevalence¹²² and progression^123,124 of diabetes. Moreover, decreased serum uromodulin is associated with higher prevalence of metabolic syndrome¹²⁵ and increased body mass index (BMI).²⁶ Circulating uromodulin levels were also inversely associated with circulating protein levels of proinflammatory adipokines chemerin and retinol-binding protein-4, especially in individuals with type 2 diabetes mellitus.¹²⁶ Circulating uromodulin concentrations were also reported to be inversely associated with markers of subclinical inflammation.¹²⁷ Considering the relationship between inflammation and metabolic disease,¹²⁸ inflammation might link circulating uromodulin to metabolic disease. A 2022 GWAS showed that the SNPs in PRKAG2 locus (encodes the γ-subunit of AMPK, which is a crucial energy sensor) is associated with elevated circulating uromodulin levels.²³ A subsequent study showed that uromodulin levels are also associated with the levels of amino and fatty acid-related metabolites in the urine, although causality was not determined.¹²⁹ These observations suggest that uromodulin might have a favorable metabolic role of uromodulin or that the metabolic state can alter uromodulin expression.

Uromodulin as a prognostic marker for AKI and CKD

Given the protective properties of uromodulin, varying levels of this protein might reflect different kidney health risk profiles. Lower urinary and circulating uromodulin levels are associated with an increased risk of acute kidney injury (AKI).^130–133 In addition, higher uromodulin levels are also associated with better AKI recovery and adaptive repair.^134–136 A 2023 study in which four subphenotypes of AKI were proposed, based on a combination of clinical and biomarker data, reported found that the subphenotype associated with the best longitudinal outcomes, including lower CKD risk, was characterized by the highest levels of uromodulin.¹³⁷ Higher uromodulin levels are also associated with lower CKD incidence^138–140 and CKD progression.^139–144 These results suggest that uromodulin levels might reflect the functional tubular response rather than just nephron mass. Moreover, studies using experimental models suggest that uromodulin is not only a biomarker of tubular health but is also protective in AKI, and in the AKI to CKD transition (discussed below).

Uromodulin levels in kidney transplantation

Following kidney allotransplantation, circulating uromodulin immediately increases from nearly undetectable values; a better recovery of circulating uromodulin levels was proposed to discriminate immediate graft function from delayed graft function.¹⁰⁷ In addition, higher pretransplant circulating uromodulin levels were associated with a lower risk of delayed graft function.¹⁴⁵ Finally, Combining uromodulin and estimated GFR (eGFR) significantly improved the ability to predict kidney graft failure.¹⁴⁶

Uromodulin levels and systemic outcomes

Higher circulating uromodulin levels are also associated with a lower risk of cardiovascular disease and all-cause mortality, even after adjustment for kidney function.^{117,124,141,147–152} These results suggest that circulating uromodulin is not just a biomarker of kidney health but can have systemic roles beyond the kidney. These data support a systemic protective role of circulating uromodulin against oxidative stress⁹¹ and vascular calcification.⁹²

Urinary non-polymeric uromodulin as a potential novel biomarker

Low urinary non-polymeric uromodulin levels might be indicative of AKI risk with potentially higher sensitivity than total urinary uromodulin.⁶⁵ These findings highlight the need for future studies that evaluate the two isoforms of urinary uromodulin separately in large studies.

Uromodulin in disease

Here we focus on the role of uromodulin in AKI, CKD and ADTKD (FIG. 4). We also discuss the importance of TAL cells in kidney disease and the association between uromodulin and hypertension.

Figure 4: Contributions of uromodulin to kidney disease.

The role of uromodulin in acute kidney injury (AKI), chronic kidney disease (CKD), and autosomal dominant tubulointerstitial kidney disease (ADTKD) is briefly summarized. The lower panel shows the therapeutic strategy targeting uromodulin. AKI induces uromodulin deficiency, and supplementation of uromodulin is a potential therapeutic approach. The role of uromodulin in CKD is complex and can be divided into two effects: non-genetic (nephron mass) and genetic (common variants). Reduced nephron mass in CKD probably causes uromodulin deficiency and seems to be harmful in CKD. However, common variants in UMOD and PDILT, are strongly associated with the risk of CKD. These variants increase uromodulin expression, but the mechanisms linking them to the enhanced disease risk are unclear. Additional effects, for example, involving other genes and independent of altered uromodulin expression, have also suggested. ADTKD is characterized by aggregation of mutant uromodulin. Clearance or knockdown of mutant uromodulin is a possible therapeutic approach. The pathogenesis of ADTKD might give insights into the mechanism of CKD.

AKI and AKI to CKD transition

AKI induces a state of uromodulin deficiency⁹ as its expression decreases after early kidney IRI^69,153 and cisplatin-induced kidney failure.¹⁵⁴ Accordingly, serum uromodulin levels decrease within 18 hours of post-surgery AKI in humans.⁹¹ Uromodulin decreases despite relative preservation of TAL cell integrity during injury,⁹⁸ suggesting an unknown dynamic response rather than loss of TAL cells. During AKI recovery, uromodulin is specifically targeted towards the interstitium (especially the basolateral region of the proximal tubule S3 segment) and circulation.⁶⁹ The aforementioned interactions with proximal tubules, neutrophils and mononuclear phagocytes^{69,70,98,99,155} suggest that uromodulin deficiency has a pro-inflammatory effect, and can exacerbate oxidative stress⁹¹. These interactions might underlie the renoprotective potential of uromodulin in AKI, and might contribute to the prevention of AKI to CKD transition.

Single-cell RNA sequencing in mouse kidney injury models revealed that Umod mRNA expression was higher in the repair model (unilateral ischemia-reperfusion injury with contralateral nephrectomy) than the atrophy model (unilateral ischemia-reperfusion injury without contralateral nephrectomy), supporting a potential role of uromodulin in adaptive repair.¹³⁶ AKI to CKD transition might be induced if uromodulin deficiency persists after AKI (for example include in cases of severe AKI requiring kidney replacement therapy, or AKI in patients with CKD). The phenotype of Umod-knockout mice in AKI to CKD transition has only been reported in the unilateral ureteral obstruction (UUO) model, which had reduced inflammation but increased KIM-1 expression and no change in fibrosis.³⁵ These data are not easily interpretable, and further analysis using different models is crucial. The protective role of uromodulin during and after AKI is strongly supported by several clinical observations demonstrating that lower uromodulin levels are associated with a higher risk of AKI and maladaptive repair (discussed earlier).

CKD

GWAS have demonstrated a robust link between uromodulin and CKD. One study reported that a SNP on a UMOD promoter, which was later shown to be associated higher UMOD expression and urinary secretion (discussed above), is associated with a higher risk of CKD.¹⁵⁶ The link between CKD (low eGFR, high serum creatinine concentration) and UMOD SNPs, including SNPs in the neighboring PDILT gene that are associated with UMOD gene expression, has been corroborated by many studies across different ethnic backgrounds.^{24,25,87,157–163} The effect size of these SNPs is often the strongest among CKD risk variants. Subsequent GWAS show that these SNPs are also associated with rapid decline of eGFR and disease progression in patients with CKD.^164–166

How this link between SNPs associated with higher uromodulin levels and higher risk of CKD can be reconciled with data suggesting that higher uromodulin expression is associated with better kidney outcomes and reduced risk of incident CKD remains unclear. The potential loss of uromodulin in CKD owing to reduced total nephron mass (as discussed above) further complicates the interpretation of these GWAS data.

The molecular mechanism by which UMOD SNPs predispose to kidney damage has not been fully determined. One study demonstrated that transgenic mice overexpressing wild-type uromodulin have age-dependent lesions in distal segments with retained kidney function.¹⁹ As the kidney lesions resembled those observed in older individuals, the researchers suggested that excessive uromodulin predisposes to kidney damage induced by age-associated co-morbidities. This hypothesis is consistent with GWAS findings showing that the association between UMOD SNPs and CKD is strongest in older individuals and in patients with comorbidities.^87,157 Determining which form of uromodulin — polymeric, non-polymeric or intracellular — is predominantly responsible for this association will be crucial. CKD-risk SNPs modulating UMOD expression might not only affect total protein abundance, but also some its polymerization and spatial distribution. One hypothesis is that high levels of intracellular uromodulin might activate membrane transporters and increase the metabolic demands of epithelial cells, which could render them more susceptible to injury.¹⁶⁷ Another potential mechanism is that increased load on the secretory pathway by increased intracellular uromodulin within TAL cells predisposes them to injury, because uromodulin is a highly abundant protein.

The impact of UMOD SNPs on genes other than UMOD⁹ is also worth investigating. Of note, some UMOD neighboring genes, such as ACSM2A/2B, are also associated with kidney function.¹⁶⁸ Therefore, the effect of UMOD SNPs on CKD risk might involve additional genes. The possibility that the link between SNPs and kidney outcomes is independent of uromodulin expression is further supported by studies showing that the effects of risk alleles on CKD incidence and progression are significantly strengthened after adjustment for uromodulin levels, even in patients without kidney disease in whom reduction in uromodulin levels is not driven by reduced nephron mass.^140,169 These hypothesis should be investigated in future studies to clarify these strong UMOD associations (Figure 4).

ADTKD-UMOD

ADTKD is an autosomal dominant genetic disease characterized by progressive tubulointerstitial damage¹⁷⁰ and is one of the most common inherited kidney diseases, after autosomal dominant polycystic kidney disease (ADPKD)¹⁷¹ and collagen IV-related diseases.¹⁷² ADTKD is characterized by slowly progressive interstitial fibrosis and kidney dysfunction without glomerular lesions. ADTKD is a relatively new disease concept that includes conventional medullary cystic kidney disease and related diseases, and is classified into subtypes according to the genes involved: UMOD, MUC1, HNF1B, REN and SEC61A1.¹⁷³ Here we focus on ADTKD-UMOD (FIG. 5) which is caused by UMOD mutations (mainly missense variants) and is one of the most frequent subtypes of this disease.¹⁷⁴

Figure 5. Pathogenesis of ADTKD-UMOD.

Heterozygous mutations in UMOD cause autosomal dominant tubulointerstitial kidney disease (ADTKD)-UMOD. Mutant uromodulin aggregates intracellularly, inducing the unfolded protein response pathway, apoptosis, inflammation, mitochondrial dysfunction and impaired autophagy. Urinary secretion of uromodulin is decreased, which reflects the intracellular aggregation of uromodulin. A potential protective role of the wild-type allele of uromodulin in the trafficking or aggregation of the mutant protein has been suggested.

In vitro^175–178 and in vivo^57,179–186 studies revealed that intracellular accumulation of mutant uromodulin is the central disease manifestation. Mutant uromodulin misfolds, is retained in the endoplasmic reticulum (ER) and aggregates, thereby inducing ER stress and the unfolded protein response (UPR) pathway, and culminating in interstitial fibrosis. The molecular mechanism that connects intracellular accumulation of uromodulin to interstitial fibrosis is well-studied but not fully elucidated. Apoptosis,^182,185 inflammation,^{57,180–182,184,186} mitochondrial dysfunction due to ER stress (secondary dysfunction)¹⁸³ and the cyclic GMP–AMP synthase (cGAS)– stimulator of interferon genes (STING) pathway¹⁷⁹ and impaired autophagy^179,182 have all been implicated.

A spatial transcriptomics analysis of ADTKD-UMOD mouse kidneys identified disease-specific cell neighborhoods around Trem2-expressing macrophages, which may have a role in the pathogenesis of ADTKD since Trem2 negatively regulates inflammation.¹⁸⁷ Urinary secretion of uromodulin is decreased both in humans and mice owing to the intracellular aggregation of uromodulin. Additionally, mutant uromodulin affects polymerization of co-expressed wild-type uromodulin.¹⁸⁸ The impact of decreased urinary polymerizing uromodulin on the pathogenesis of ADTKD is unknown. Circulating uromodulin levels in patients with ADTKD-UMOD, although much less studied than urinary uromodulin levels, were reportedly lower than in reference patients with CKD.¹⁸⁹ A 2023 report showed that deletion of wild-type Umod in heterozygous Umod-mutant ADTKD-UMOD mice increased aggregation of uromodulin and exacerbated the phenotype⁵⁷. This observation highlights a potential protective role of wild-type uromodulin in intracellular homeostasis, including the trafficking or aggregation of mutant protein. The existence of atypical ADTKD-UMOD, which mainly involves glomerular lesions, has been reported¹⁹⁰, but its pathogenesis remains unclear.

One study reported that an intermediate-effect UMOD variant (T62P), which is present in ~1/1,000 individuals with European ancestry, is associated with higher risk of developing CKD. Human UMOD T62 showed an ADTKD-like trafficking and maturation defect, but the phenotype was less severe than that of canonical ADTKD-causing mutant in HEK293 and MDCK cells.¹⁹¹ This intermediate variant raises the possibility that CKD and ADTKD might be in a broad disease spectrum, and that ADTKD research could provide insights into the pathogenesis of CKD.

Emerging importance of TAL cells in kidney disease

The involvement of uromodulin in abroad spectrum of kidney disease raises questions about the role of TAL and early DCT cells on global kidney function. TAL–proximal tubule crosstalk (inflammation and oxidative stress) and TAL–immune cell crosstalk might explain this effect. In a transgenic mouse model, targeted damage to TAL cells was sufficient to induce severe AKI.¹⁹² Moreover, a subsequent multimodal single-cell and spatial analysis of healthy and diseased human kidneys from the Kidney Precision Medicine Project, in which we participated, identified adaptive (successful or maladaptive repair) and degenerative (damaged or stressed) TAL cell subtypes. This work demonstrated the unique and independent association of an injured TAL signature with kidney disease progression.¹⁹³ These findings suggest that TAL injury itself can affect global kidney function. Further multi-omics, single-cell and spatial analyses might elucidate the significance of TAL cell injury, as well as TAL cell crosstalk with proximal tubule and immune cells, in kidney disease.

Hypertension

Uromodulin has a key role in the pathogenesis of hypertension, independently of kidney function (reviewed in¹⁹⁴). Briefly, uromodulin handles sodium absorption by interacting with sodium transporters and thus modulates blood pressure. A UMOD SNP that leads to elevated uromodulin expression, is also associated with a higher risk of hypertension¹⁹⁵ and a potential causal relationship was reported using Mendelian randomization.¹⁶⁷ Consistent with this mechanism, Umod transgenic mice, which mimic the risk variants, had hyper-phosphorylation of NKCC2 and salt-sensitive hypertension.¹⁹ Of note, circulating uromodulin levels were inversely associated with the vasoconstrictive prohormone pro-endothelin-1 (CT-proET-1) and arterial hypertension, suggesting a potential additional role of uromodulin in hypertension.¹⁹⁶

Therapeutic opportunities to target uromodulin

Based on the mounting evidence discussed above, regulating uromodulin expression might be a promising therapeutic strategy for a broad spectrum of kidney and systemic diseases. Here, we discuss four potential strategies for modulating uromodulin.

Supplementation with non-polymeric uromodulin

Uromodulin supplementation might be beneficial to accelerate kidney repair in AKI⁷⁰. As discussed above, basolateral release of uromodulin into the interstitium and circulation have a key role in the protective effects of uromodulin in AKI. Further studies of truncated monomeric uromodulin supplementation in different AKI models and at varying time courses and doses are underway to investigate potential clinical applications. Patients with CKD are at high risk of AKI and probably have less uromodulin owing to decreased nephron mass. Therefore, uromodulin supplementation in patients with CKD at high-risk of AKI (for example, owing to surgery, infections or exposure to nephrotoxins) might have a protective effect. Uromodulin overexpression also ameliorated vascular calcification in cholecalciferol overloaded mice.⁹²

Complexity of altering uromodulin expression in CKD

As discussed above, the role of uromodulin in CKD is complex, and the potential benefits of interventions targeting uromodulin will be context-dependent. When CKD causes uromodulin deficiency through reduced total nephron mass, uromodulin supplementation might be beneficial. For patients with genetic variants associated with increased uromodulin expression, reduction of uromodulin expression might be a potential intervention, guided by genotypes and uromodulin levels. However, considering the multiple protective effects of uromodulin including protection against AKI, and the uncertainty regarding which form contributes to CKD susceptibility, strategies to reduce the overall uromodulin expression might pose risks. Further research on the dynamics and roles of each form of uromodulin in CKD-risk SNP carriers is needed to propose a selective and efficacious approach with minimal off-target complications.

Down-regulating mutant uromodulin in ADTKD-UMOD

Although currently no specific treatment for ADTKD-UMOD is available, some studies have revealed potential therapeutic opportunities. Initially, based on the findings that ADTKD-UMOD is an ER-stress-mediated disease, the chemical chaperone sodium 4-phenylbutyrate was tested but found not to be effective in an ADTKD-UMOD mouse model.¹⁹⁷ This result led to further investigation of the pathogenesis and alternative therapeutic approaches. TNF inhibition reduced apoptosis and alleviated disease progression in ADTKD-UMOD cellular and mouse models.¹⁸² Another promising and evolving approach is to reduce the aggregation of mutant uromodulin. Autophagy activation by mammalian target of rapamycin complex 1 (mTORC1) inhibition^57,182 and starvation⁵⁷ decreased aggregated uromodulin in vitro. Moreover, BRD4780, which is a small molecule targeting the cargo receptor TMED9 involved in vesicular targeting to the lysosome, cleared mutant uromodulin and alleviated ER stress in vitro.¹⁹⁸ Another study demonstrated that overexpression of mesencephalic astrocyte-derived neurotrophic factor (MANF), which is an ER protein, cleared mutant uromodulin by activating autophagy and mitophagy, and mitigated kidney injury in vitro and in vivo.¹⁷⁹ The clinical feasibility of these approaches will depend on their ability to specifically degrade mutant uromodulin given the potential side effects of degrading other essential proteins. Gene-editing and gene-silencing of mutant Umod alleles might be another promising approach.

Targeting NKCC2 in hypertension

An ongoing clinical trial is using a pharmacogenomics-based approach to test the efficacy of loop diuretics, which inhibit NKCC2, on patients with uncontrolled hypertension and who are genotyped for known UMOD SNPs.¹⁹⁹

Conclusions

Since its discovery as a urinary protein, the functional significance of uromodulin has been rather enigmatic. Technological innovation and intensive research, especially in the last two decades, have substantially expanded our understanding of uromodulin in health and disease. Urinary uromodulin protects against urinary tract infections and kidney stones, and maintains the homeostasis of the urinary space. New studies have also reported various protective roles of uromodulin in the interstitium, circulation and intracellularly. The presence of multiple forms might explain the versatile effects of uromodulin. In addition to polymeric uromodulin, which predominates in urine, non-polymerizing uromodulin can also be detected in urine and circulation. Additional mechanisms might also regulate the localization and retention of uromodulin at specific cellular sites — further investigation is required, which should also clarify the contributions of uromodulin to kidney and systemic immunity.

The association of uromodulin with common kidney diseases including AKI, CKD, ADTKD and hypertension has accelerated uromodulin research. Uromodulin is protective against AKI, and AKI induces a state of uromodulin deficiency. Supplementation with non-polymerizing uromodulin might improve the course of AKI and possibly protect from a transition to CKD, but further research is required to investigate the therapeutic potential of this approach. The association between uromodulin and CKD is robust but complex, and how SNPs in UMOD and PDILT predispose individuals to CKD remains a challenging question. Whether this effect depends on a specific form of uromodulin expression needs to be clarified. Finally, ADTKD is characterized by intracellular aggregation of mutant uromodulin and clearance of the mutant protein is a promising therapeutic approach. In addition, insights from ADTKD research might contribute to a better understanding of the association between uromodulin and CKD. Future uromodulin research should uncover uromodulin biology further and assess its potential for clinical applications (Box 1).

Box 1. Future directions of uromodulin research.

1. Structural analyses, including comparison of polymeric and non-polymeric forms using high-resolution technologies, should clarify the role of uromodulin in health and disease:

   • Form-specific analyses in biology and biomarker research should elucidate biological mechanisms and inform clinical applicability.
   • Single-cell and spatial analysis will clarify the regulation of uromodulin, as well as its involvement in the pathology of kidney disease, including the role of UMOD-expressing cells and their neighboring cells.

2. Identification of the intracellular mechanisms governing the processing and secretion of uromodulin

3. Determination of the impact of sexual dimorphism on uromodulin expression and implications for sex differences in kidney disease.

4. Investigation of potential clinical applications:

   • The efficacy of uromodulin supplementation for AKI and other uromodulin-related disease should be examined further.
   • Clearance of mutant uromodulin, as well as gene therapy, might be beneficial in ADTKD.
   • Elucidation, through experimental and clinical evidence, of the molecular mechanism by which UMOD single-nucleotide polymorphisms (SNPs) predispose to kidney damage is needed and should inform novel therapeutic approaches.

Key points.

• Most urinary uromodulin undergoes polymerization into filaments and has important roles in maintaining urinary homeostasis. This polymerization occurs on the apical membrane and is highly organized.

• Non-polymeric uromodulin, an increasingly recognized form, has distinct kidney and systemic roles. Recognizing the specific form and site of action is crucial to understanding the function of uromodulin.

• Acute kidney injury (AKI) is characterized by uromodulin deficiency. Non-polymerizing uromodulin supplementation might improve the course of AKI and prevent the transition to chronic kidney disease (CKD).

• Single-nucleotide polymorphisms in UMOD and PDILT are strongly linked to the risk and progression of CKD. Whether this effect depends on expression of a specific form of uromodulin remains to be clarified.

• Retention and aggregation of mutant uromodulin in the endoplasmic reticulum causes autosomal dominant tubulointerstitial kidney disease. Clearance of mutant uromodulin might be a promising therapeutic approach.