Uromodulin, Inflammasomes, and Pyroptosis
Authors: Maria Dolores Sanchez-Niño, Ana B. Sanz, Alberto Ortiz
Journal: Journal of the American Society of Nephrology (2012)

As a kidney-specific protein, uromodulin has fascinated nephrologists for decades.1 Initially known as Tamm-Horsfall protein, uromodulin is secreted by distal tubules into the urine, where it is the most abundant protein under physiologic conditions. Such an abundant protein surely has a key function. In the 1980s, uromodulin was described as an immunosuppressive protein that bound and neutralized IL-1β and other cytokines.1 Consistent with this observation, studies in null mice demonstrated a protective role of uromodulin against kidney ischemia-reperfusion injury.1 Uromodulin also prevents renal stone formation and, counterintuitively for an immunosuppressive protein, urinary tract infections.1 In this regard, uromodulin may directly interact with uropathogens. Mutations in the human gene encoding uromodulin lead to autosomal dominant CKD. Recently, genome-wide association studies identified the uromodulin gene as a risk factor for CKD and hypertension. However, in a recurrent nephrologist's nightmare, uromodulin drops its gentle appearance to attack the kidney. Although uromodulin is mostly confined to the intratubular space in the normal kidney, conditions that damage nephron integrity or cause tubular cell stress may increase uromodulin release into the interstitial space and facilitate access to the circulation. In the 1980s anti-Tamm-Horsfall antibodies were thought to contribute to chronic pyelonephritis.1 Cell culture evidence suggests that extratubular uromodulin may promote inflammation in myeloid cells, for example, by activating dendritic cells through engagement of Toll-like receptor-4.1,2 A proinflammatory role of uromodulin is further supported by the observation that intravenous administration of uromodulin leads to systemic inflammation.2 However, there is little in vivo evidence of a proinflammatory effect of local uromodulin during kidney injury. A central conundrum of uromodulin biology is how to reconcile its proinflammatory actions on cultured leukocytes or after systemic administration with the nephroprotective effect observed in vivo during kidney injury. In this issue of JASN, Darisipudi et al. contribute to a better understanding of uromodulin function during kidney injury by reporting that uromodulin nanoparticles activate the NLRP3 inflammasome in human monocytes, leading to the release of IL-1β and cell death.3 Priming by other proinflammatory stimuli increases pro-IL-1β availability and further contributes to increased IL-1β release.3 NLRP3 inflammasome activation and IL-1β release require the phagocytic uptake of uromodulin particles into lysosomes, cathepsin leakage, oxidative stress, and potassium efflux from the cell,3 similar to observations for other nanoparticles or crystal activators of the NLRP3 inflammasome.4,5 Inflammasomes are a set of intracellular protein complexes that in response to microbial products (pathogen-associated molecular patterns [PAMPs]) or metabolic stress (damage-associated molecular patterns [DAMPs]) enable autocatalytic activation of inflammatory caspases, promoting the release of leaderless cytokines such as IL-1β and IL-18, alarmins, and lipid mediators, inducing pyroptosis.4,6 Inflammasomes are composed of platform proteins, adaptor proteins (ASCs), and effector proteins (inflammatory caspases, such as caspase-1). The inflammasome containing the NLRP3 platform protein (NLRP3 inflammasome) has been well characterized primarily in leukocytes. Darisipudi et al. suggest that the findings of inflammasome activation by uromodulin may provide a molecular explanation for divergent roles of uromodulin as a nephroprotector when confined inside tubules, and uromodulin as an endogenous, proinflammatory DAMP when dislocated in the renal interstitium.3 However, as with any significant cell biology advance, new questions arise. The first question refers to the molecular determinants of the lack of tubular cell activation by uromodulin located inside tubules. Tubular cells express components of the inflammasome: Both NLRP3 and ASC are highly expressed in human and murine renal tubular epithelium.7 Pro-IL-1β, NLRP3, ASC and procaspase-1 mRNA are increased compared with controls in the tubular areas of murine GN, while pro-IL-1β and NLRP3 but not ASC are increased in the flow-through, interstitial fraction.8 Tubular cells are reported to be the main site of IL-18 protein expression during ischemia-reperfusion renal injury,9 and kidney cell deficiency in IL-18 protects from ischemia-reperfusion injury.10 Recent experiments in chimeric Nlrp3 null mice showed NLRP3 biologic function in both hematopoietic and renal compartments during renal injury induced by ureteral obstruction and cycloheximide/TNF-α–induced apoptosis are reduced in cultured Nlrp3−/− tubular cells, confirming a biologic role for Nlrp3 in renal tubules.11 However, Nlrp3 may have an inflammasome-independent role in tubular cells. Absence of Nlrp3 in tubular epithelium, but not absence of Asc or caspase-1, protects from renal injury, inflammation, and tubular cell apoptosis in renal ischemia-reperfusion injury, whereas absence of Nlrp3 in bone marrow cells does not protect.7 Thus, tubular cells may express components of the inflammasome. However, levels of expression appear to be lower than in professional phagocytes. Darisipudi et al. suggest that the inability of renal parenchymal cells to secrete IL-1β may explain why uromodulin remains immunologically inert inside the luminal compartment of the urinary tract.3 They performed experiments but do not show data in this respect. This information is consistent with prior data, in which low pro-IL-1β and pro-IL-18 levels of expression in cultured tubular cells prevents the study of the role of NLRP3 in cytokine processing.11 In addition, tubular cells might lack efficient uromodulin phagocytosis. The second question relates to the apparent discrepancy between the observed anti-inflammatory effect of uromodulin during kidney injury in uromodulin null mice and cell culture data in which uromodulin activates the inflammasome. The hypothesis of Darisipudi et al. that the presence of uromodulin in the interstitial space elicits renal inflammation and triggers kidney damage is at odds the in vivo data from null mice.1,3 Is there any alternative hypothesis? Uromodulin triggers cell death in macrophages.3 The characteristics of uromodulin-triggered cell death are reminiscent of pyroptosis.4,12 Thus, it is associated with cell lysis, inflammasome activation, and IL-1β release. Pyroptosis is a relatively novel concept and little more than 100 papers had been published on the subject by August 2012. The term pyroptosis was originally introduced to describe a program of cell death in macrophages characterized by cell lysis induced by bacterial infection that requires caspase-1 activation and is associated with release of pyrogenic ILs such as IL-1β and IL-1.4,12 However, pyroptosis is independent from IL-1β and IL-18. As in apoptosis, pyroptosis is caspase dependent, but differs from apoptosis in the loss of cell membrane integrity and release of proinflammatory cytokines.13 Most studies characterize pyroptosis in macrophages in response to intracellular bacteria. In this system, pyroptosis contributes to bacterial clearance by the innate immune system. Thus, bacteria released from macrophages by pyroptosis are killed by neutrophils.14 In this regard, the hypothesis that uromodulin-induced pyroptosis may contribute to the observed antibacterial effects of uromodulin in vivo should be tested.1 Bacterial products and sterile nanoparticles can also trigger pyroptosis.15,16 Consistent with the hypothesis advanced by Darisipudi et al. of an injurious role of inflammasome activation by uromodulin, toxin-induced pyroptosis may promote tissue injury, as shown for the lung.15 However, the role of pyroptosis in kidney injury or in sterile inflammation has not been well characterized. The demonstration that uromodulin may induce cell death with characteristics of pyroptosis may help understand the regulation of pyroptosis and the study of its role in kidney injury. In this regard, different kinds of phenotypically polarized macrophages have been described and are generally termed proinflammatory M1 or classically activated, and anti-inflammatory M2 or alternatively activated.17 Macrophages are able to dynamically and reversibly switch from one state to another.18 In M1 macrophages, the NLRP3 inflammasome is activated by diverse stimuli. By contrast, M2 macrophages downregulate caspase-1 and IL-1β and fail to activate the inflammasome and release IL-1β in response to inflammasome agonists.18 The susceptibility of M2 macrophages to pyroptosis has not been characterized but would be expected to be low. Furthermore, activation of the inflammasome and IL-1β release induced by the DAMP and canonical agonist ATP in M1 macrophages ceases over time. This is related to changes in the macrophage phenotype associated with a paradoxical inhibition by ATP of the activation by other stimuli of an intact NLRP3 inflammasome, thus effectively promoting an M2 phenotype.18 The possibility that uromodulin, by promoting M1 macrophage pyroptosis, shifts the balance toward the M2 anti-inflammatory phenotype should also be explored. In this regard, there is little information on macrophage subsets in uromodulin null mice during kidney injury. Thus, by promoting IL-1β release, uromodulin might favor inflammation. However, by promoting pyroptosis, it may disrupt the balance between different macrophage functional subsets. As is the case during infection, in which macrophage pyroptotic death improves antibacterial defenses, the hypothesis that macrophage death may also contribute to nephroprotection should be explored. In conclusion, for the first time, a kidney-specific protein has been shown to promote inflammasome activation and a mode of cell death compatible with pyroptosis in monocytic cells. Darisipudi et al. hypothesize that this observation could explain putative proinflammatory effects of uromodulin when it reaches the tubulointerstitial space. Alternative hypotheses should be explored that are more in line with the in vivo observation that uromodulin deficiency aggravates ischemia-reperfusion renal injury and predisposes to urinary tract infections. These hypotheses include a potential role of uromodulin-induced pyroptosis in modulating the number of proinflammatory macrophages and regulating the antibacterial response. Disclosures None. Grant support is provided by ISCII and FEDER (PS09/00447), Sociedad Española de Nefrologia (ISCIII-RETIC REDinREN/RD06/0016), and the Comunidad de Madrid (CIFRA/S2010/BMD-2378). Salary support is provided by FIS Sara Borrel to M.D.S.N. and A.B.S., as well as by the Programa Intensificación Actividad Investigadora (ISCIII/Agencia Laín-Entralgo/CM) to A.O.