A Uromodulin Mutation Drives Autoimmunity and Kidney Mononuclear Phagocyte Endoplasmic Reticulum Stress.

We identified a family with a UMOD gene mutation (C106F) resulting in glomerular inflammation and complement deposition. To determine if the observed phenotype is due to immune system activation by mutant uromodulin, a mouse strain with a homologous cysteine to phenylalanine mutation (C105F) in the UMOD gene was generated using CRISPR-Cas9 gene editing and the effect of this mutation on mononuclear phagocytic cells was examined. Mutant mice developed high levels of intracellular and secreted aggregated uromodulin, resulting in anti-uromodulin antibodies and circulating uromodulin containing immune complexes with glomerular deposition and kidney fibrosis with aging. F4/80+ and CD11c+ kidney cells phagocytize uromodulin. Differential gene expression analysis by RNA sequencing of F4/80+ phagocytic cells revealed activation of the activating transcription factor 5 (ATF5)-mediated stress response pathway in mutant mice. Phagocytosis of mutant uromodulin by cultured dendritic cells resulted in activation of the endoplasmic reticulum stress response pathway and markers of cell inactivation, an effect not seen with wild-type protein. Mutant mice demonstrate a twofold increase in T-regulatory cells, consistent with induction of immune tolerance, resulting in decreased inflammatory response and improved tissue repair following ischemia-reperfusion injury. The C105F mutation results in autoantibodies against aggregated misfolded protein with immune complex formation and kidney fibrosis. Aggregated uromodulin may induce dendritic cell tolerance following phagocytosis through an unfolded protein/endoplasmic reticulum stress response pathway, resulting in decreased inflammation following tissue injury.

Extratubular Polymerized Uromodulin Induces Leukocyte Recruitment and Inflammation In Vivo.

Uromodulin (UMOD) is produced and secreted by tubular epithelial cells. Secreted UMOD polymerizes (pUMOD) in the tubular lumen, where it regulates salt transport and protects the kidney from bacteria and stone formation. Under various pathological conditions, pUMOD accumulates within the tubular lumen and reaches extratubular sites where it may interact with renal interstitial cells. Here, we investigated the potential of extratubular pUMOD to act as a damage associated molecular pattern (DAMP) molecule thereby creating local inflammation. We found that intrascrotal and intraperitoneal injection of pUMOD induced leukocyte recruitment in vivo and led to TNF-α secretion by F4/80 positive macrophages. Additionally, pUMOD directly affected vascular permeability and increased neutrophil extravasation independent of macrophage-released TNF-α. Interestingly, pUMOD displayed no chemotactic properties on neutrophils, did not directly activate ß2 integrins and did not upregulate adhesion molecules on endothelial cells. In obstructed neonatal murine kidneys, we observed extratubular UMOD accumulation in the renal interstitium with tubular atrophy and leukocyte infiltrates. Finally, we found extratubular UMOD deposits associated with peritubular leukocyte infiltration in kidneys from patients with inflammatory kidney diseases. Taken together, we identified extratubular pUMOD as a strong inducer of leukocyte recruitment, underlining its critical role in mounting an inflammatory response in various kidneys pathologies.

Tamm-Horsfall Protein Protects the Urinary Tract against Candida albicans.

Urinary tract infections (UTIs) caused by the human fungal pathogen Candida albicans and related species are prevalent in hospitalized patients, especially those on antibiotic therapy, with indwelling catheters, or with predisposing conditions such as diabetes or immunodeficiency. Understanding of key host defenses against Candida UTI is critical for developing effective treatment strategies. Tamm-Horsfall glycoprotein (THP) is the most abundant urine protein, with multiple roles in renal physiology and bladder protection. THP protects against bacterial UTI by blocking bacterial adherence to the bladder epithelium, but its role in defense against fungal pathogens is not yet described. Here we demonstrate that THP restricts colonization of the urinary tract by C. albicans THP binds to C. albicans hyphae, but not the yeast form, in a manner dependent on fungal expression of the Als3 adhesion glycoprotein. THP directly blocks C. albicans adherence to bladder epithelial cells in vitro, and THP-deficient mice display increased fungal burden in a C. albicans UTI model. This work outlines a previously unknown role for THP as an essential component for host immune defense against fungal urinary tract infection.

Improving body fluid identification in forensic trace evidence-construction of an immunochromatographic test array to rapidly detect up to five body fluids simultaneously.

Body fluid identification is a substantial part of forensic trace analyses. The correct determination of the origin of a biological stain may give valuable information regarding the circumstances of a crime. A simple way to detect a body fluid in a stain is the use of immunochromatographic strip tests. They are easy to use, user-independent, quick, and cheap. Currently, however, it is only possible to analyze one body fluid at a time, requiring the analyst to make previous, possibly subjective, assumptions on the body fluid at hand. Also, identification of mixed body fluids requires the use of several tests, which results in additional sample and time consumption. To combine a simple approach with the possibility to simultaneously detect several body fluids, we constructed a combined immunochromatographic strip test array based on commercially available tests. The array rapidly detects up to five body fluids with a single analysis, and allowing for subsequent DNA extraction from the same material. With this test it was possible to identify the components of a mixture, the test was easily incorporated into standard laboratory work, and its sensitivity and specificity were shown to be comparable to those of conventional strip tests.

Complement activation and kidney injury molecule-1-associated proximal tubule injury in severe preeclampsia.

Kidney injury with proteinuria is a characteristic feature of preeclampsia, yet the nature of injury in specific regions of the nephron is incompletely understood. Our study aimed to use existing urinary biomarkers to describe the pattern of kidney injury and proteinuria in pregnancies affected by severe preeclampsia. We performed a case-control study of pregnant women from Brigham and Women's Hospital from 2012 to 2013. We matched cases of severe preeclampsia (n=25) 1:1 by parity and gestational age to 2 control groups with and without chronic hypertension. Urinary levels of kidney injury molecule-1 and complement components (C3a, C5a, and C5b-9) were measured by enzyme-linked immunosorbent assay, and other markers (albumin, ß2 microglobulin, cystatin C, epithelial growth factor, neutrophil gelatinase-associated lipocalin, osteopontin, and uromodulin) were measured simultaneously with a multiplex electrochemiluminescence assay. Median values between groups were compared with the Wilcoxon signed-rank test and correlations with Spearman correlation coefficient. Analysis of urinary markers revealed higher excretion of albumin and kidney injury molecule-1 and lower excretion of neutrophil gelatinase-associated lipocalin and epithelial growth factor in severe preeclampsia compared with chronic hypertension and healthy controls. Among subjects with severe preeclampsia, urinary excretion of complement activation products correlated most closely with kidney injury molecule-1, a specific marker of proximal tubule injury (C5a: r=0.60; P=0.001; and C5b-9: r=0.75; P<0.0001). Taken together, we describe a pattern of kidney injury in severe preeclampsia that is characterized by glomerular impairment and complement-mediated inflammation and injury, possibly localized to the proximal tubule in association with kidney injury molecule-1.

The signaling pathway of uromodulin and its role in kidney diseases.

The uromodulin (UMOD) is a glycoprotein expressed exclusively by renal tubular cells lining the thick ascending limb of the loop of Henle. UMOD acts as a regulatory protein in health and in various conditions. For kidney diseases, its role remains elusive. On one hand, UMOD plays a role in binding and excretion of various potentially injurious products from the tubular fluid. On the other hand, chronic kidney disease is associated with higher serum levels of UMOD. Signaling pathways might be very important in the pathogenesis of kidney diseases. We performed this review to provide a relatively complete signaling pathway flowchart for UMOD to the investigators who were interested in the role of UMOD in the pathogenesis of kidney diseases. Here, we reviewed the signal transduction pathway of UMOD and its role in the pathogenesis of kidney diseases.

Uromodulin triggers IL-1ß-dependent innate immunity via the NLRP3 inflammasome.

Uromodulin/Tamm-Horsfall protein is not immunostimulatory in the tubular lumen, but through unknown mechanisms it can activate dendritic cells and promote inflammation in the renal interstitium. Here, we noted that uromodulin isolated from human urine aggregates to large, irregular clumps with a crystal-like ultrastructure. These uromodulin nanoparticles activated isolated human monocytes to express costimulatory molecules and to secrete the mature proinflammatory cytokines, including IL-1ß. Full release of IL-1ß in response to uromodulin depended on priming of pro-IL-1ß expression by Toll-like receptors, TNF-α, or IL-1α. In addition, uromodulin-induced secretion of mature IL-1ß depended on the NLRP3 inflammasome, its linker molecule ASC, and pro-IL-1ß cleavage by caspase-1. Activation of NLRP3 required phagocytosis of uromodulin particles into lysosomes, cathepsin leakage, oxidative stress, and potassium efflux from the cell. Taken together, these data suggest that uromodulin is a NLRP3 agonist handled by antigen-presenting cells as an immunostimulatory nanoparticle. Thus, in the presence of tubular damage that exposes the renal interstitium, uromodulin becomes an endogenous danger signal. The inability of renal parenchymal cells to secrete IL-1ß may explain why uromodulin remains immunologically inert inside the luminal compartment of the urinary tract.

Specificity, sensitivity, and operability of RSID™-urine for forensic identification of urine: comparison with ELISA for Tamm-Horsfall protein.

In this study, the specificity, sensitivity, and operability of RSID™-Urine, a new immunochromatographic test for urine identification, was evaluated and compared with ELISA detection of Tamm-Horsfall protein (THP). Urine was successfully identified among other body fluids using RSID™-Urine and ELISA detection of THP. The detection limit of RSID™-Urine equated to 0.5 µL of urine; although the sensitivity of RSID™-Urine may be lower than that of ELISA detection of THP, it is thought to be sufficient for application to casework samples. However, results from RSID™-Urine must be interpreted with caution when the sample may have been contaminated with blood or vaginal fluid, because this might inhibit urine detection. The RSID™-Urine assay can be performed in just 15 min by dropping the extracted sample onto the test cassette. Therefore, RSID™-Urine should be an effective tool for the forensic identification of urine, in addition to ELISA detection of THP.

Tamm-Horsfall protein regulates circulating and renal cytokines by affecting glomerular filtration rate and acting as a urinary cytokine trap.

Although few organ systems play a more important role than the kidneys in cytokine catabolism, the mechanism(s) regulating this pivotal physiological function and how its deficiency affects systemic cytokine homeostasis remain unclear. Here we show that elimination of Tamm-Horsfall protein (THP) expression from mouse kidneys caused a marked elevation of circulating IFN-γ, IL1α, TNF-α, IL6, CXCL1, and IL13. Accompanying this were enlarged spleens with prominent white-pulp macrophage infiltration. Lipopolysaccharide (LPS) exacerbated the increase of serum cytokines without a corresponding increase in their urinary excretion in THP knock-out (KO) mice. This, along with the rise of serum cystatin C and the reduced inulin and creatinine clearance from the circulation, suggested that diminished glomerular filtration may contribute to reduced cytokine clearance in THP KO mice both at the baseline and under stress. Unlike wild-type mice where renal and urinary cytokines formed specific in vivo complexes with THP, this "trapping" effect was absent in THP KO mice, thus explaining why cytokine signaling pathways were activated in renal epithelial cells in such mice. Our study provides new evidence implicating an important role of THP in influencing cytokine clearance and acting as a decoy receptor for urinary cytokines. Based on these and other data, we present a unifying model that underscores the role of THP as a major regulator of renal and systemic immunity.