RESUMEN
Uromodulin [Tamm-Horsfall protein (THP)] is a glycoprotein uniquely produced in the kidney. It is released by cells of the thick ascending limbs apically in the urine and basolaterally in the renal interstitium and systemic circulation. Processing of mature urinary THP, which polymerizes into supramolecular filaments, requires cleavage of an external hydrophobic patch (EHP) at the COOH-terminus. However, THP in the circulation is not polymerized, and it remains unclear if nonaggregated forms of THP exist natively in the urine. We propose that an alternative processing path, which retains the EHP domain, can lead to a nonpolymerizing form of THP. We generated an antibody that specifically recognizes THP with retained EHP (THP + EHP) and established its presence in the urine in a nonpolymerized native state. Proteomic characterization of urinary THP + EHP revealed its COOH-terminus ending at F617. In the human kidney, THP + EHP was detected in thick ascending limb cells and less strongly in the renal parenchyma. Using immunoprecipitation followed by proteomic sequencing and immunoblot analysis, we then demonstrated that serum THP has also retained EHP. In a small cohort of patients at risk for acute kidney injury, admission urinary THP + EHP was significantly lower in patients who subsequently developed acute kidney injury during hospitalization. Our findings uncover novel insights into uromodulin biology by establishing the presence of an alternative path for cellular processing, which could explain the release of nonpolymerizing THP in the circulation. Larger studies are needed to establish the utility of urinary THP + EHP as a sensitive biomarker of kidney health and susceptibility to injury.NEW & NOTEWORTHY In this work, we discovered and characterized a novel form of uromodulin that does not polymerize because it retains an external hydrophobic patch at the COOH-terminus. These findings establish an alternative form of cellular processing of this protein and elucidate new aspects of its biology. We also provide evidence suggesting that measuring urinary nonpolymerizing uromodulin could be a promising assay to assess the risk of acute kidney injury.
Asunto(s)
Lesión Renal Aguda , Riñón , Proteómica , Uromodulina , Lesión Renal Aguda/metabolismo , Humanos , Riñón/metabolismo , Uromodulina/química , Uromodulina/orinaRESUMEN
Sepsis is a significant cause of mortality in hospitalized patients. Concomitant development of acute kidney injury (AKI) increases sepsis mortality through unclear mechanisms. Although electrolyte disturbances and toxic metabolite buildup during AKI could be important, it is possible that the kidney produces a protective molecule lost during sepsis with AKI. We have previously demonstrated that systemic Tamm-Horsfall protein (THP; uromodulin), a kidney-derived protein with immunomodulatory properties, falls in AKI. Using a mouse sepsis model without severe kidney injury, we showed that the kidney increases circulating THP by enhancing the basolateral release of THP from medullary thick ascending limb cells. In patients with sepsis, changes in circulating THP were positively associated with a critical illness. THP was also found de novo in injured lungs. Genetic ablation of THP in mice led to increased mortality and bacterial burden during sepsis. Consistent with the increased bacterial burden, the presence of THP in vitro and in vivo led macrophages and monocytes to upregulate a transcriptional program promoting cell migration, phagocytosis, and chemotaxis, and treatment of macrophages with purified THP increases phagocytosis. Rescue of septic THP-/- mice with exogenous systemic THP improved survival. Together, these findings suggest that through releasing THP, the kidney modulates the immune response in sepsis by enhancing mononuclear phagocyte function, and systemic THP has therapeutic potential in sepsis.NEW & NOTEWORTHY Specific therapies to improve outcomes in sepsis with kidney injury have been limited by an unclear understanding of how kidney injury increases sepsis mortality. Here, we identified Tamm-Horsfall protein, known to protect in ischemic acute kidney injury, as protective in preclinical sepsis models. Tamm-Horsfall protein also increased in clinical sepsis without severe kidney injury and concentrated in injured organs. Further study could lead to novel sepsis therapeutics.
Asunto(s)
Lesión Renal Aguda , Sepsis , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/prevención & control , Animales , Modelos Animales de Enfermedad , Riñón/metabolismo , Sepsis/complicaciones , Sepsis/metabolismo , Uromodulina/genética , Uromodulina/metabolismoRESUMEN
Autosomal dominant tubulointerstitial disease (ADTKD) is a dominantly inherited progressive nonglomerular disease. Several factors, such as a nonspecific clinical presentation and relative rarity, impede the phenotyping of ADTKD into clinically relevant subtypes and impair the appropriate implementation of genetic testing. The study by Olinger et al. describes the largest multicenter ADTKD cohort, which is likely to become a key resource. The authors also provide a new clinical tool that could guide diagnosis and genetic testing.
Asunto(s)
Nefritis Intersticial , Riñón Poliquístico Autosómico Dominante , Pruebas Genéticas , Humanos , Mucina-1/genética , Mutación , Nefritis Intersticial/diagnóstico , Nefritis Intersticial/genética , Riñón Poliquístico Autosómico Dominante/diagnóstico , Riñón Poliquístico Autosómico Dominante/genéticaRESUMEN
Protozoan parasites, including the apicomplexan pathogens Plasmodium falciparum (which causes malaria) and Toxoplasma gondii (which causes toxoplasmosis), infect millions of people worldwide and represent major human disease burdens. Despite their prevalence, therapeutic strategies to treat infections caused by these parasites remain limited and are threatened by the emergence of drug resistance, highlighting the need for the identification of novel drug targets. Recently, homologues of the core autophagy proteins, including Atg8 and Atg3, were identified in many protozoan parasites. Importantly, components of the Atg8 conjugation system that facilitate the lipidation of Atg8 are required for both canonical and parasite-specific functions and are essential for parasite viability. Structural characterization of the P. falciparum Atg3-Atg8 (PfAtg3-Atg8) interaction has led to the identification of compounds that block this interaction. Additionally, many of these compounds inhibit P. falciparum growth in vitro, demonstrating the viability of this pathway as a drug target. Given the essential role of the Atg8 lipidation pathway in Toxoplasma, we sought to determine whether three PfAtg3-Atg8 interaction inhibitors identified in the Medicines for Malaria Venture Malaria Box exerted a similar inhibitory effect in Toxoplasma While all three inhibitors blocked Toxoplasma replication in vitro at submicromolar concentrations, they did not inhibit T. gondii Atg8 (TgAtg8) lipidation. Rather, high concentrations of two of these compounds induced TgAtg8 lipidation and fragmentation of the parasite mitochondrion, similar to the effects seen following starvation and monensin-induced autophagy. Additionally, we report that one of the PfAtg3-Atg8 interaction inhibitors induces Toxoplasma egress and provide evidence that this is mediated by an increase in intracellular calcium in response to drug treatment.
Asunto(s)
Antiprotozoarios/farmacología , Proteínas Relacionadas con la Autofagia/antagonistas & inhibidores , Plasmodium falciparum/efectos de los fármacos , Isoformas de Proteínas/antagonistas & inhibidores , Proteínas Protozoarias/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Toxoplasma/efectos de los fármacos , Secuencia de Aminoácidos , Antiprotozoarios/química , Autofagia/efectos de los fármacos , Proteínas Relacionadas con la Autofagia/química , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Sitios de Unión , Replicación del ADN/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/parasitología , Expresión Génica , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Simulación del Acoplamiento Molecular , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Unión Proteica/efectos de los fármacos , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Bibliotecas de Moléculas Pequeñas/química , Especificidad de la Especie , Relación Estructura-Actividad , Toxoplasma/genética , Toxoplasma/crecimiento & desarrollo , Toxoplasma/metabolismoRESUMEN
Toxoplasma gondii is an obligate intracellular apicomplexan parasite with high seroprevalence in humans. Repeated lytic cycles of invasion, replication, and egress drive both the propagation and the virulence of this parasite. Key steps in this cycle, including invasion and egress, depend on tightly regulated calcium fluxes and, although many of the calcium-dependent effectors have been identified, the factors that detect and regulate the calcium fluxes are mostly unknown. To address this knowledge gap, we used a forward genetic approach to isolate mutants resistant to extracellular exposure to the calcium ionophore A23187. Through whole genome sequencing and complementation, we have determined that a nonsense mutation in a previously uncharacterised protein is responsible for the ionophore resistance of one of the mutants. The complete loss of this protein recapitulates the resistance phenotype and importantly shows defects in calcium regulation and in the timing of egress. The affected protein, GRA41, localises to the dense granules and is secreted into the parasitophorous vacuole where it associates with the tubulovesicular network. Our findings support a connection between the tubulovesicular network and ion homeostasis within the parasite, and thus a novel role for the vacuole of this important pathogen.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/crecimiento & desarrollo , Toxoplasmosis/parasitología , Calcimicina/farmacología , Ionóforos de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Vesículas Citoplasmáticas/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de la Membrana/genética , Proteínas Protozoarias/genética , Toxoplasma/genética , Toxoplasma/metabolismo , Toxoplasmosis/transmisión , Vacuolas/metabolismoRESUMEN
The essential base excision repair protein, apurinic/apyrimidinic endonuclease 1 (APE1), plays an important role in redox regulation in cells and is currently targeted for the development of cancer therapeutics. One compound that binds APE1 directly is (E)-3-[2-(5,6-dimethoxy-3-methyl-1,4-benzoquinonyl)]-2-nonylpropenoic acid (E3330). Here, we revisit the mechanism by which this negatively charged compound interacts with APE1 and inhibits its redox activity. At high concentrations (millimolar), E3330 interacts with two regions in the endonuclease active site of APE1, as mapped by hydrogen-deuterium exchange mass spectrometry. However, this interaction lowers the melting temperature of APE1, which is consistent with a loss of structure in APE1, as measured by both differential scanning fluorimetry and circular dichroism. These results are consistent with other findings that E3330 concentrations of >100 µM are required to inhibit APE1's endonuclease activity. To determine the role of E3330's negatively charged carboxylate in redox inhibition, we converted the carboxylate to an amide by synthesizing (E)-2-[(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)methylene]-N-methoxy-undecanamide (E3330-amide), a novel uncharged derivative. E3330-amide has no effect on the melting temperature of APE1, suggesting that it does not interact with the fully folded protein. However, E3330-amide inhibits APE1's redox activity in in vitro electrophoretic mobility shift redox and cell-based transactivation assays, producing IC(50) values (8.5 and 7 µM) lower than those produced with E3330 (20 and 55 µM, respectively). Thus, E3330's negatively charged carboxylate is not required for redox inhibition. Collectively, our results provide additional support for a mechanism of redox inhibition involving interaction of E3330 or E3330-amide with partially unfolded APE1.
Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa/antagonistas & inhibidores , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Fluorometría , Espectrometría de Masas , Modelos Moleculares , Oxidación-Reducción , Activación TranscripcionalRESUMEN
BACKGROUND: Uromodulin is a protein made only by the kidney and released in urine, circulating in polymerizing and nonpolymerizing forms. This protein's multiple functions include inhibition of stone formation in the urine. The physiological determinants of uromodulin production are incompletely understood. METHODS: We investigated changes in uromodulin levels and key factors governing its production and release in urine and serum. We performed an experiment to determine whether water loading, a common intervention to prevent stone formation, will alter the rate of uromodulin production. During a 2-day period, 17 stone forming participants and 14 control participants were subjected to water loading (day 1) and normal fluid intake (day 2). Uromodulin levels were measured on timed hourly collections in urine and plasma during the period of the study. RESULTS: Water loading increased urinary uromodulin secretion (33±4 versus 10±4 µ g/min at baseline, P < 0.0001) in stone formers and control participants. Despite high urine volumes, most participants maintained relatively stable urinary uromodulin concentrations. Native Western blots for polymerizing and nonpolymerizing uromodulin suggest that polymerizing uromodulin was the predominant form at higher urinary flow volumes. Urine flow rates and sodium excretion were significant correlates of urinary uromodulin production. Water loading did not affect serum uromodulin levels, which were also not associated with urinary uromodulin. CONCLUSIONS: Water loading increases the secretion of polymerizing urinary uromodulin. This increased secretion reduces the variability of urinary uromodulin concentrations despite high urine volumes. Serum uromodulin levels were not affected by this treatment.
Asunto(s)
Calcio , Cálculos Renales , Humanos , Uromodulina , Calcio/orina , Cálculos Renales/orina , Agua , Riñón/metabolismoRESUMEN
Kidney stone disease causes significant morbidity and increases health care utilization. In this work, we decipher the cellular and molecular niche of the human renal papilla in patients with calcium oxalate (CaOx) stone disease and healthy subjects. In addition to identifying cell types important in papillary physiology, we characterize collecting duct cell subtypes and an undifferentiated epithelial cell type that was more prevalent in stone patients. Despite the focal nature of mineral deposition in nephrolithiasis, we uncover a global injury signature characterized by immune activation, oxidative stress and extracellular matrix remodeling. We also identify the association of MMP7 and MMP9 expression with stone disease and mineral deposition, respectively. MMP7 and MMP9 are significantly increased in the urine of patients with CaOx stone disease, and their levels correlate with disease activity. Our results define the spatial molecular landscape and specific pathways contributing to stone-mediated injury in the human papilla and identify associated urinary biomarkers.
Asunto(s)
Cálculos Renales , Médula Renal , Humanos , Médula Renal/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasa 7 de la Matriz , Oxalato de Calcio/metabolismo , Transcriptoma , Cálculos Renales/genética , Cálculos Renales/metabolismoRESUMEN
Tamm-Horsfall protein (THP) is produced exclusively by the kidney, where it is released into both the urine and the circulation. Although the primary form of circulating THP is nonpolymerizing, urinary THP exists as a mix of polymerizing and nonpolymerizing forms. Urinary THP has been shown to play roles in such disparate processes as prevention of urinary tract infections and kidney stone formation, along with the regulation of multiple ion channels within the kidney. The generation of THP knockout mouse models has allowed the investigation of these phenomena and shown a prospective role for circulating THP in ischemia-reperfusion acute kidney injury as well as sepsis. Recent studies have suggested that THP is protective in ischemic injury owing to its inhibition of oxidative stress via the calcium channel transient receptor potential cation channel, subfamily M, member 2 t(TRPM2), and protection in sepsis is at least partially due to THP's promotion of macrophage function.
Asunto(s)
Lesión Renal Aguda , Canales Catiónicos TRPM , Animales , Ratones , Humanos , Uromodulina/metabolismo , Riñón/metabolismo , Lesión Renal Aguda/metabolismo , Ratones Noqueados , Estrés Oxidativo , Canales Catiónicos TRPM/metabolismoRESUMEN
Uromodulin (or Tamm-Horsfall protein) is a glycoprotein uniquely produced in the kidney by tubular cells of the thick ascending limb of the loop of Henle and early distal tubules. This protein exhibits bidirectional secretion in the urine and in the renal interstitium and circulation. The role of this protein in maintaining renal and systemic homeostasis is becoming increasingly appreciated. Furthermore, perturbations of its functions may play a role in various diseases affecting the kidney and distant organs. In this review, we will discuss important advances in understanding its biology, highlighting the recent discoveries of its secretion and differential precursor processing that generates 2 forms: (1) a highly polymerizing form that is apically excreted in the urine and generates filaments and (2) a nonpolymerizing form that retains a polymerization inhibitory pro-peptide and is released basolaterally in the kidney interstitium and circulation, but can also be found in the urine. We will also discuss factors regulating its production and release, taking into account its intricate physiology, and propose best practices to report its levels. We also discuss breaking advances in its role in hypertension, acute kidney injury and progression to chronic disease, immunomodulation and regulating renal and systemic oxidative stress. We anticipate that this work will be a great resource for researchers and clinicians. This review will highlight the importance of defining what regulates the 2 forms of uromodulin, so that modulation of uromodulin levels and function could become a novel tool in our therapeutic armamentarium against kidney disease.
Asunto(s)
Lesión Renal Aguda , Hipertensión , Humanos , Uromodulina/metabolismo , Riñón/metabolismo , Lesión Renal Aguda/metabolismo , Hipertensión/metabolismo , BiologíaRESUMEN
BACKGROUND: Deceased-donor kidneys experience extensive injury, activating adaptive and maladaptive pathways therefore impacting graft function. We evaluated urinary donor uromodulin (UMOD) and osteopontin (OPN) in recipient graft outcomes. METHODS: Primary outcomes: all-cause graft failure (GF) and death-censored GF (dcGF). Secondary outcomes: delayed graft function (DGF) and 6-month estimated glomerular filtration rate (eGFR). We randomly divided our cohort of deceased donors and recipients into training and test datasets. We internally validated associations between donor urine UMOD and OPN at time of procurement, with our primary outcomes. The direction of association between biomarkers and GF contrasted. Subsequently, we evaluated UMOD:OPN ratio with all outcomes. To understand these mechanisms, we examined the effect of UMOD on expression of major histocompatibility complex II in mouse macrophages. RESULTS: Doubling of UMOD increased dcGF risk (adjusted hazard ratio [aHR], 1.1; 95% confidence interval [CI], 1.02-1.2), whereas OPN decreased dcGF risk (aHR, 0.94; 95% CI, 0.88-1). UMOD:OPN ratio ≤3 strengthened the association, with reduced dcGF risk (aHR, 0.57; 0.41-0.80) with similar associations for GF, and in the test dataset. A ratio ≤3 was also associated with lower DGF (aOR, 0.73; 95% CI, 0.60-0.89) and higher 6-month eGFR (adjusted ß coefficient, 3.19; 95% CI, 1.28-5.11). UMOD increased major histocompatibility complex II expression elucidating a possible mechanism behind UMOD's association with GF. CONCLUSIONS: UMOD:OPN ratio ≤3 was protective, with lower risk of DGF, higher 6-month eGFR, and improved graft survival. This ratio may supplement existing strategies for evaluating kidney quality and allocation decisions regarding deceased-donor kidney transplantation.
Asunto(s)
Funcionamiento Retardado del Injerto/etiología , Tasa de Filtración Glomerular , Supervivencia de Injerto , Trasplante de Riñón/efectos adversos , Riñón/cirugía , Osteopontina/orina , Donantes de Tejidos , Uromodulina/orina , Adulto , Anciano , Animales , Biomarcadores/orina , Células Cultivadas , Funcionamiento Retardado del Injerto/mortalidad , Funcionamiento Retardado del Injerto/fisiopatología , Femenino , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Riñón/fisiopatología , Trasplante de Riñón/mortalidad , Macrófagos/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Estudios Prospectivos , Medición de Riesgo , Factores de Riesgo , Factores de Tiempo , Estados UnidosRESUMEN
High serum concentrations of kidney-derived protein uromodulin [Tamm-Horsfall protein (THP)] have recently been shown to be independently associated with low mortality in both older adults and cardiac patients, but the underlying mechanism remains unclear. Here, we show that THP inhibits the generation of reactive oxygen species (ROS) both in the kidney and systemically. Consistent with this experimental data, the concentration of circulating THP in patients with surgery-induced acute kidney injury (AKI) correlated with systemic oxidative damage. THP in the serum dropped after AKI and was associated with an increase in systemic ROS. The increase in oxidant injury correlated with postsurgical mortality and need for dialysis. Mechanistically, THP inhibited the activation of the transient receptor potential cation channel, subfamily M, member 2 (TRPM2) channel. Furthermore, inhibition of TRPM2 in vivo in a mouse model mitigated the systemic increase in ROS during AKI and THP deficiency. Our results suggest that THP is a key regulator of systemic oxidative stress by suppressing TRPM2 activity, and our findings might help explain how circulating THP deficiency is linked with poor outcomes and increased mortality.