Structure of the decoy module of human glycoprotein 2 and uromodulin and its interaction with bacterial adhesin FimH.

Glycoprotein 2 (GP2) and uromodulin (UMOD) filaments protect against gastrointestinal and urinary tract infections by acting as decoys for bacterial fimbrial lectin FimH. By combining AlphaFold2 predictions with X-ray crystallography and cryo-EM, we show that these proteins contain a bipartite decoy module whose new fold presents the high-mannose glycan recognized by FimH. The structure rationalizes UMOD mutations associated with kidney diseases and visualizes a key epitope implicated in cast nephropathy.

Acute kidney injury in a mouse model of meningococcal disease.

INTRODUCTION: Meningococcal disease is associated with high mortality. When acute kidney injury (AKI) occurs in patients with severe meningococcal disease, it is typically attributable to sepsis, although meningococcal disease and lipopolysaccharide release are rarely investigated. Therefore, we evaluated renal tissue in a mouse model of meningococcal disease. METHODS: Female BALB/c mice were induced to AKI by meningococcal challenge. Markers of renal function were evaluated in infected and control mice. RESULTS: In the infected mice, serum concentrations of tumor necrosis factor alpha, interferon gamma, interleukins (IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-12), and granulocyte-macrophage colony-stimulating factor were elevated, as was renal interstitial infiltration with lymphocytes and neutrophils (p < 0.01 for the latter). Histological analysis showed meningococcal microcolonies in the renal interstitium, without acute tubular necrosis. Infected mice also showed elevated renal expression of toll-like receptor 2, toll-like receptor 4, and Tamm-Horsfall protein. The expression of factors in the intrinsic pathway of apoptosis was equal to or lower than that observed in the control mice. Urinary sodium and potassium were also lower in infected mice, probably due to a tubular defect. CONCLUSION: Our findings corroborate those of other studies of AKI in sepsis. To our knowledge, this is the first time that meningococci have been identified in renal interstitium and that the resulting apoptosis and inflammation have been evaluated. However, additional studies are needed in order to elucidate the mechanisms involved.

Using machine learning to study protein-protein interactions: From the uromodulin polymer to egg zona pellucida filaments.

Neural network-based models for protein structure prediction have recently reached near-experimental accuracy and are fast becoming a powerful tool in the arsenal of biologists. As suggested by initial studies using RoseTTAFold or the ColabFold implementation of AlphaFold2, a particularly interesting future development will be the optimization of these computational methods to also routinely yield high-confidence predictions of protein-protein interactions. Here I use AlphaFold2 and ColabFold to investigate the activation and polymerization of uromodulin (UMOD)/Tamm-Horsfall protein, a zona pellucida (ZP) module-containing protein whose precursor and filamentous structures have been previously determined experimentally by X-ray crystallography and cryo-EM, respectively. Despite having no knowledge of the UMOD polymer structure (coordinates for which were neither used for model training nor as template), AlphaFold2/ColabFold are able to recapitulate a crucial conformational change underlying UMOD polymerization, as well as the general organization of protein subunits within the resulting filament. This surprising result is achieved by simply deleting from the input sequence a stretch of residues that correspond to a polymerization-inhibiting C-terminal propeptide. By mimicking in silico the activating effect of propeptide dissociation triggered by site-specific proteolysis of the protein precursor, this example has implications for the assembly of egg coat proteins and the many other molecules that also contain a ZP module. Most importantly, it shows the potential of exploiting machine learning not only to accurately predict the structures of individual proteins or complexes, but also to carry out computational experiments replicating specific molecular events.

Clinically Viable Assay for Monitoring Uromodulin Glycosylation.

Uromodulin, also known as the Tamm-Horsfall protein or THP, is the most abundant protein excreted in human urine. It is associated with the progression of kidney diseases; therefore, changes in the glycosylation profile of this protein could serve as a potential biomarker for kidney health. The typical glycomics analysis approaches used to quantify uromodulin glycosylation involve time-consuming and tedious glycoprotein isolation and labeling steps, which limit their utility in clinical glycomics assays, where sample throughput is important. Herein, we introduce a radically simplified sample preparation workflow, with direct ESI-MS analysis, enabling the quantification of N-linked glycans that originate from uromodulin. The method omits any glycan labeling steps but includes steps to reduce the salt content of the samples, thereby minimizing ion suppression. The method is effective for quantifying subtle glycosylation differences of uromodulin samples derived from different biological states. As a proof of concept, glycosylation from samples that differ by pregnancy status were shown to be differentiable.

Identification and detection of protein markers to differentiate between forensically relevant body fluids.

The identification of body fluids at a crime scene is an important aspect of forensic casework analysis, being a source for investigative leads and contributing to case evidence. Yet, current methods for the forensic identification of body fluids suffer from several limitations, ranging from poor sensitivity and specificity, to sample destruction and interference with subsequent DNA analysis. Moreover, current identification assays target only one body fluid at the time. Besides being inefficient in terms of time, money and sample consumption, poor identification methods can also negatively influence the outcome of a (court) case. In this study, eleven potential protein biomarkers and antibodies were selected and assessed on their suitability for serving as identification markers, as a first step towards the development of a new multiplex protein-based body fluid identification assay relying on antigen-antibody interactions. Performing antibody-based dot blot assays, the specificity of the biomarkers for their target body fluids was evaluated, and biomarker detection was studied in diluted, mixed, aged and simulated casework samples. Hereby, nine out of eleven markers were identified as promising biomarkers to identify blood, semen, saliva, urine and sweat. With the identification of these targets and detection antibodies, a major step forward has been taken towards the development of a highly sensitive and specific, fast and non-labour-intensive protein-based body fluid identification assay, suitable for on-site analysis and able to test for multiple body fluids in a single reaction.

Fate of Neutrophils during the Recovery Phase of Ischemia/Reperfusion Induced Acute Kidney Injury.

Effective clearance of inflammatory cells is required for resolution of inflammation. Here, we show in vivo evidence that apoptosis and reverse transendothelial migration (rTEM) are important mechanisms in eliminating neutrophils and facilitating recovery following ischemia/reperfusion injury (IRI) of the kidney. The clearance of neutrophils was delayed in the Bax knockout (KO)(BM) → wild-type (WT) chimera in which bone marrow derived cells are partially resistant to apoptosis, compared to WT(BM) → WT mice. These mice also showed delayed functional, histological recovery, increased tissue cytokines, and accelerated fibrosis. The circulating intercellular adhesion molecule-1 (ICAM-1)⁺ Gr-1⁺ neutrophils displaying rTEM phenotype increased during the recovery phase and blockade of junctional adhesion molecule-C (JAM-C), a negative regulator of rTEM, resulted in an increase in circulating ICAM-1⁺ neutrophils, faster resolution of inflammation and recovery. The presence of Tamm-Horsfall protein (THP) in circulating ICAM-1⁺ neutrophils could suggest that they are derived from injured kidneys. In conclusion, we suggest that apoptosis and rTEM are critically involved in the clearance mechanisms of neutrophils during the recovery phase of IRI.

[Uromodulin gene polymorphisms in patients with cast nephropathy in multiple myeloma]. / Polimorfizmy gena uromodulina u bol'nykh kast-nefropatiei pri mnozhestvennoi mielome.

AIM: To investigate the nature of mutations in exons 4 and 5 of the uromodulin (UM) gene, including in the area encoding the domain of 8 cysteines (D8C), in patients with multiple myeloma (MM) with the secretion of monoclonal light chains (LC) in cast nephropathy (CN) and without kidney injury. SUBJECTS AND METHODS: The investigation enrolled 24 patients in MM remission, who were observed to have monoclonal LC secretion at onset. Group 1 included 14 patients with CN; Group 2 consisted of 10 patients with normal renal function (a comparison group). The compared groups did not differ in the number of serum and urinary monoclonal LCs. Genomic DNA was extracted from the peripheral blood samples of patients. The nucleotide sequence of exons 4 and 5 of the UM gene was determined by the Sanger method. RESULTS: No differences were found in the frequency of polymorphisms depending on the severity of kidney injury. The missense mutation p.142R>R/Q in the UM gene, which had not been previously described, was discovered. CONCLUSION: The patients with MM were not found to have statistically significant differences in the frequency and nature of polymorphisms of exons 4 and 5 in the UM gene, including in the area encoding D8C, in CN without kidney injury.

Uromodulin: a new biomarker of fetal renal function?

INTRODUCTION: Obstructive uropathies are main diseases affecting the fetus. Early diagnosis allows to establish the appropriate therapy to minimize the risk of damage to kidney function at birth. Biochemical markers have been used to predict the prognosis of renal function in fetuses. Uromodulin, also known by Tamm-Horsfall protein (THP) is exclusively produced in the kidneys and in normal conditions is the protein excreted in larger amounts in human urine. It plays important roles in kidneys and urinary tract. Also it participates in ion transport processes, interact with various components of the immune system and has a role in defense against urinary tract infections. Moreover, this protein was proved to be a good marker of renal function in adult patients with several renal diseases. OBJECTIVE: To evaluate if uromodulin is produced and eliminated by the kidneys during fetal life by analyzing fetal urine and amniotic fluid and to establish correlation with biochemical parameter of renal function already used in Fetal Medicine Center at the Clinic Hospital of UFMG (CEMEFE/HC). METHODS: Between 2013 and 2015, were selected 29 fetuses with indication of invasive tests for fetal diagnosis in monitoring at the CEMEFE/HC. RESULTS: The determination of uromodulin was possible and measurable in all samples and showed statistically significant correlation with the osmolarity. CONCLUSION: There was a tendency of lower levels of Uromodulin values in fetuses with severe renal impairment prenatally. Thus, high levels of this protein in fetal amniotic fluid or fetal urine dosages possibly mean kidney function preserved.

Uromodulin: a new biomarker of fetal renal function? / Uromodulina: um novo biomarcador de função renal fetal?

Abstract Introduction: Obstructive uropathies are main diseases affecting the fetus. Early diagnosis allows to establish the appropriate therapy to minimize the risk of damage to kidney function at birth. Biochemical markers have been used to predict the prognosis of renal function in fetuses. Uromodulin, also known by Tamm-Horsfall protein (THP) is exclusively produced in the kidneys and in normal conditions is the protein excreted in larger amounts in human urine. It plays important roles in kidneys and urinary tract. Also it participates in ion transport processes, interact with various components of the immune system and has a role in defense against urinary tract infections. Moreover, this protein was proved to be a good marker of renal function in adult patients with several renal diseases. Objective: To evaluate if uromodulin is produced and eliminated by the kidneys during fetal life by analyzing fetal urine and amniotic fluid and to establish correlation with biochemical parameter of renal function already used in Fetal Medicine Center at the Clinic Hospital of UFMG (CEMEFE/HC). Methods: Between 2013 and 2015, were selected 29 fetuses with indication of invasive tests for fetal diagnosis in monitoring at the CEMEFE/HC. Results: The determination of uromodulin was possible and measurable in all samples and showed statistically significant correlation with the osmolarity. Conclusion: There was a tendency of lower levels of Uromodulin values in fetuses with severe renal impairment prenatally. Thus, high levels of this protein in fetal amniotic fluid or fetal urine dosages possibly mean kidney function preserved.

Resumo Introdução: Uropatias obstrutivas estão entre as principais doenças que acometem o feto. O diagnóstico precoce destas doenças permite estabelecer a terapêutica adequada, visando minimizar os riscos de danos à função renal no nascimento. Os marcadores bioquímicos têm sido utilizados na predição do prognóstico da função renal em fetos. A uromodulina, também chamada de proteína de Tamm-Horsfall (THP), é produzida exclusivamente nos rins, e em condições normais, é a proteína excretada em maior volume na urina humana. Ela desempenha importantes funções nos rins e trato urinário. Participa dos processos de transporte de íons, interage com vários componentes do sistema imunológico e possui papel na defesa contra infecções do trato urinário. Além disso, se mostrou um bom biomarcador de função renal em adultos portadores de diversas doenças renais. Objetivos: Avaliar se a uromodulina é produzida e eliminada pelos rins durante a vida fetal através da análise de urina fetal e líquido amniótico, além de estabelecer correlação com o parâmetro bioquímico de função renal já utilizado no Centro de Medicina Fetal do Hospital das Clínicas da UFMG (CEMEFE/HC). Métodos: Entre 2013 e 2015, foram selecionados 29 fetos com indicação de exames invasivos para diagnóstico fetal em acompanhamento no CEMEFE/HC. Resultados: A dosagem da uromodulina foi possível e quantificável em todas as amostras e mostrou correlação significativa com a osmolaridade. Conclusão: A uromodulina mostrou uma tendência em apresentar valores reduzidos em fetos com grave comprometimento renal no pré-natal. Assim, valores elevados desta proteína em dosagens de urina fetal ou líquido amniótico podem significar uma função renal preservada.

Development and characterization of a pseudo multiple reaction monitoring method for the quantification of human uromodulin in urine.

BACKGROUND: Uromodulin is the most abundant protein in healthy human urine. Recently it has been suggested as a specific biomarker of renal tubular damage. We have developed a novel pseudo multiple reaction monitoring (pseudo MRM) for the protein's quantification in human urine. RESULTS: Selection of two peptides allowed quantification of uromodulin in human urine. The pseudo MRM quantified uromodulin in healthy individuals between 21 and 1344 nM and in autosomal dominant tubulointerstitial kidney disease-UMOD patients between 2 and 25 nM. CONCLUSION: The pseudo MRM allows greater confidence in assay specificity than traditional MRM methods and quantified uromodulin at concentrations higher than achievable by ELISA. Differences in urinary uromodulin concentration related to the rs4293393 promoter variant in the UMOD gene was confirmed. This method will be used to further investigate uromodulin as a biomarker of renal injury.

Smaller caliber renal arteries are a novel feature of uromodulin-associated kidney disease.

Hyperuricemia is very common in industrialized countries and known to promote vascular smooth muscle cell proliferation. Juvenile hyperuricemia is a hallmark of uromodulin-associated kidney disease characterized by progressive interstitial renal fibrosis leading to end-stage renal disease within decades. Here we describe a member of a Polish-German family with a history of familial background of chronic kidney disease, hyperuricemia, and gout. This patient had hypertension because of bilateral small renal arteries, hyperuricemia, and chronic kidney disease. Clinical and molecular studies were subsequently performed in 39 family members, which included a physical examination, Duplex ultrasound of the kidneys, laboratory tests for renal function, and urine analysis. In eight family members contrast-enhanced renal artery imaging by computed tomography-angiography or magnetic resonance imaging was conducted and showed that bilateral non-arteriosclerotic small caliber renal arteries were associated with hyperuricemia and chronic kidney disease. Of the 26 family members who underwent genotyping, 11 possessed the P236R mutation (c.707C>G) of the uromodulin gene. All family members with a small caliber renal artery carried the uromodulin P236R mutation. Statistical analysis showed a strong correlation between reduced renal artery lumen and decreased estimated glomerular filtration rate. Thus, bilateral small caliber renal arteries are a new clinical phenotype associated with an uromodulin mutation.

Mass spectrometric study of stone matrix proteins of human bladder stones.

OBJECTIVE: To evaluate the mechanisms of bladder uric acid stone (BUAS) formation by analyzing BUAS stone matrix proteins, with mass spectrometry (MS). MATERIALS AND METHODS: Stone matrix proteins were extracted from 5 pure BUASs. The obtained proteins were analyzed with reverse phase liquid chromatography-tandem MS. The acquired data were investigated against a Swiss Prot human protein database, using Matrix Science Mascot. The identified proteins were submitted to UniProtKB website for gene ontology analysis to define their correlation. They were also submitted to Metacore platform and Kyoto Encyclopedia of Genes and Genomes website for pathway analysis. MS-determined protein expressions were validated by immunoblot. RESULTS: The liquid chromatography-tandem MS analysis identified 58-226 proteins in the 5 BUASs (450 proteins). Metacore software analysis suggests that inflammation might play an important role for BUAS formation. The analysis of endogenous metabolic pathways revealed that these proteins were categorized into glycerophospholipid or glycosphingolipid biosynthesis. Four of 5 identified proteins selected for validation, including uromodulin, S100P, Histone 4, and nucleophosmin, can be validated in the immunoblot data. CONCLUSION: Our results suggest that inflammatory process and lipid metabolism might play a role in the formation of BUAS. Whether these inflammatory responses are the etiology of stone formation or whether they result from local damage by stone irritation is uncertain.

Uromodulin and α(1)-antitrypsin urinary peptide analysis to differentiate glomerular kidney diseases.

BACKGROUND/AIMS: Glomerular kidney disease (GKD) is suspected in patients based on proteinuria, but its diagnosis relies primarily on renal biopsy. We used urine peptide profiling as a noninvasive means to link GKD-associated changes to each glomerular entity. METHODS: Urinary peptide profiles of 60 biopsy-proven glomerular patients and 14 controls were analyzed by combining magnetic bead peptide enrichment, MALDI-TOF MS analysis, and ClinProTools v2.0 to select differential peptides. Tentative identification of the differential peptides was carried out by HPLC-MS/MS. RESULTS: The HPLC-MS/MS results suggest that uromodulin (UMOD; m/z: 1682, 1898 and 1913) and α(1)-antitrypsin (A1AT; m/z: 1945, 2392 and 2505) are differentially expressed urinary peptides that distinguish between GKD patients and healthy subjects. Low UMOD and high A1AT peptide abundance was observed in 80-92% of patients with GKD. Proliferative forms of GKD were distinguished from nonproliferative forms, based on a combination of UMOD and A1AT peptides. Nonproliferative forms correlated with higher A1AT peptide levels - focal segmental glomerulosclerosis was linked more closely to high levels of the m/z 1945 peptide than minimal change disease. CONCLUSION: We describe a workflow - urinary peptide profiling coupled with histological findings - that can be used to distinguish GKD accurately and noninvasively, particularly its nonproliferative forms.

Expression of heme oxygenase-1 in thick ascending loop of henle attenuates angiotensin II-dependent hypertension.

Kidney-specific induction of heme oxygenase-1 (HO-1) attenuates the development of angiotensin II (Ang II) -dependent hypertension, but the relative contribution of vascular versus tubular induction of HO-1 is unknown. To determine the specific contribution of thick ascending loop of Henle (TALH) -derived HO-1, we generated a transgenic mouse in which the uromodulin promoter controlled expression of human HO-1. Quantitative RT-PCR and confocal microscopy confirmed successful localization of the HO-1 transgene to TALH tubule segments. Medullary HO activity, but not cortical HO activity, was significantly higher in transgenic mice than control mice. Enhanced TALH HO-1 attenuated the hypertension induced by Ang II delivered by an osmotic minipump for 10 days (139 ± 3 versus 153 ±2 mmHg in the transgenic and control mice, respectively; P<0.05). The lower blood pressure in transgenic mice associated with a 60% decrease in medullary NKCC2 transporter expression determined by Western blot. Transgenic mice also exhibited a 36% decrease in ouabain-sensitive sodium reabsorption and a significantly attenuated response to furosemide in isolated TALH segments. In summary, these results show that increased levels of HO-1 in the TALH can lower blood pressure by a mechanism that may include alterations in NKCC2-dependent sodium reabsorption.

[Lympho-venous shunts and thromboids in urinary obstruction]. / Lymphovenöse Shunts und Thromboide bei Harnstauungsniere.

This case of urinary obstruction with short anamnesis illuminates the early tissue reaction to urine extravasation, especially the Tamm-Horsfall protein. The latter becomes organized in the manner of a foreign body substance and drains via ectatic renal lymph vessels and reopened lymphovenous shunts into the intrarenal venous system. Polypoid formations (thromboids) with or without vessel obliteration appear to result in a clinical course of severe renal impairment.

Uromodulin exclusion list improves urinary exosomal protein identification.

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

Immunohistochemical identification of kidney nephron segments in the dog, rat, mouse, and cynomolgus monkey.

Kidney is a major target organ in preclinical studies. In recent years, intense research has been undertaken to characterize novel renal toxicity biomarkers. In this context, we studied nephron segment specific antibodies against aquaporin-1 (AQP-1), α-glutathione-S-transferase (alpha-GST), Tamm-Horsfall protein (TH), calbindin-D(28K) (CalD), and aquaporin-2 (AQP-2), using an immunoperoxidase method on formalin-fixed paraffin-embedded kidney tissues of dogs, rats, mice, and Cynomolgus monkeys. AQP-1 was specific for proximal tubules and thin descending limbs of Henle's loops and AQP-2 for connecting and collecting ducts in dogs, rats, mice, and Cynomolgus monkeys. Alpha-GST stained the straight part of proximal tubules in dogs and proximal convoluted tubule and straight part of proximal tubules in rats. TH was specific for thick ascending limbs of Henle's loops in mice, rats, dogs, and Cynomolgus monkeys and stained additionally scattered cells in cortical connecting/collecting ducts of dogs. CalD was found in distal convoluted tubules and cortical connecting and collecting ducts of dogs, rats, and mice and in distal convoluted tubules, connecting ducts, and cortical and medullary collecting ducts of Cynomolgus monkey. This panel of antibodies may be a helpful tool to identify renal tubules by light microscopy in preclinical studies and to validate new biomarkers of renal toxicity.

Uromodulin and chronic kidney disease.

Uromodulin (Tamm-Horsfall protein) is produced in the kidney by cells of the thick ascending limb and distal tubule. Recent genetic studies suggest a role of uromodulin in chronic kidney disease. Mutations in the UMOD gene cause uromodulin storage disease. They code for amino acid substitutions that lead to misfolding of the molecule and its retention in the endoplasmic reticulum. Single nucleotide polymorphisms in the region of the UMOD gene have been shown to be associated with chronic kidney disease and reduced glomerular filtration rate. These polymorphisms affect uromodulin concentration in the urine, and lower genetically determined urinary uromodulin concentrations seem to protect against renal disease. Chronic kidney disease is associated with higher serum levels of uromodulin. From animal experiments and human studies it is hypothesized that uromodulin entering the renal interstitium either by basolateral secretion or urinary back-leakage in damaged tubuli interacts with and stimulates cells of the immune system and thereby causes inflammation and progression of chronic kidney disease.