ABSTRACT: We show that resident macrophages accumulate in cilia mutant mice prior to cyst formation and that inhibition of resident macrophage accumulation reduces cystic kidney disease. Overall design: We analyzed gene expression of R2a (CD11c+) and R2b (CD11c-) resident macrophages isolated from adult, wild type mice. In these experiments, we isolated RNA from 6-8 mice and pooled the RNA together prior to sequencing.
Project description:BACKGROUND:Mutations affecting cilia proteins have an established role in renal cyst formation. In mice, the rate of cystogenesis is influenced by the age at which cilia dysfunction occurs and whether the kidney has been injured. Disruption of cilia function before postnatal day 12-14 results in rapid cyst formation; however, cyst formation is slower when cilia dysfunction is induced after postnatal day 14. Rapid cyst formation can also be induced in conditional adult cilia mutant mice by introducing renal injury. Previous studies indicate that macrophages are involved in cyst formation, however the specific role and type of macrophages responsible has not been clarified. METHODS:We analyzed resident macrophage number and subtypes during postnatal renal maturation and after renal injury in control and conditional Ift88 cilia mutant mice. We also used a pharmacological inhibitor of resident macrophage proliferation and accumulation to determine the importance of these cells during rapid cyst formation. RESULTS:Our data show that renal resident macrophages undergo a phenotypic switch from R2b (CD11clo) to R2a (CD11chi) during postnatal renal maturation. The timing of this switch correlates with the period in which cyst formation transitions from rapid to slow following induction of cilia dysfunction. Renal injury induces the reaccumulation of juvenile-like R2b resident macrophages in cilia mutant mice and restores rapid cystogenesis. Loss of primary cilia in injured conditional Ift88 mice results in enhanced epithelial production of membrane-bound CSF1, a cytokine that promotes resident macrophage proliferation. Inhibiting CSF1/CSF1-receptor signaling with a CSF1R kinase inhibitor reduces resident macrophage proliferation, R2b resident macrophage accumulation, and renal cyst formation in two mouse models of cystic disease. CONCLUSIONS:These data uncover an important pathogenic role for resident macrophages during rapid cyst progression.
Project description:The assembly of primary cilia is dependent on intraflagellar transport (IFT), which mediates the bidirectional movement of proteins between the base and tip of the cilium. In mice, congenic mutations disrupting genes required for IFT (e.g., Tg737 or the IFT kinesin Kif3a) are embryonic lethal, whereas kidney-specific disruption of IFT results in severe, rapidly progressing cystic pathology. Although the function of primary cilia in most tissues is unknown, in the kidney they are mechanosenstive organelles that detect fluid flow through the tubule lumen. The loss of this flow-induced signaling pathway is thought to be a major contributing factor to cyst formation. Recent data also suggest that there is a connection between ciliary dysfunction and obesity as evidenced by the discovery that proteins associated with human obesity syndromes such as Alström and Bardet-Biedl localize to this organelle. To more directly assess the importance of cilia in postnatal life, we utilized conditional alleles of two ciliogenic genes (Tg737 and Kif3a) to systemically induce cilia loss in adults. Surprisingly, the cystic kidney pathology in these mutants is dependent on the time at which cilia loss was induced, suggesting that cyst formation is not simply caused by impaired mechanosensation. In addition to the cystic pathology, the conditional cilia mutant mice become obese, are hyperphagic, and have elevated levels of serum insulin, glucose, and leptin. We further defined where in the body cilia are required for normal energy homeostasis by disrupting cilia on neurons throughout the central nervous system and on pro-opiomelanocortin-expressing cells in the hypothalamus, both of which resulted in obesity. These data establish that neuronal cilia function in a pathway regulating satiety responses.
Project description:Zebrafish are an attractive model for studying the earliest cellular defects occurring during renal cyst formation because its kidney (the pronephros) is simple and genes that cause cystic kidney diseases (CKD) in humans, cause pronephric dilations in zebrafish. By comparing phenotypes in three different mutants, locke, swt and kurly, we find that dilations occur prior to 48 hpf in the medial tubules, a location similar to where cysts form in some mammalian diseases. We demonstrate that the first observable phenotypes associated with dilation include cilia motility and luminal remodeling defects. Importantly, we show that some phenotypes common to human CKD, such as an increased number of cells, are secondary consequences of dilation. Despite having differences in cilia motility, locke, swt and kurly share similar cystic phenotypes, suggesting that they function in a common pathway. To begin to understand the molecular mechanisms involved in cyst formation, we have cloned the swt mutation and find that it encodes a novel leucine rich repeat containing protein (LRRC50), which is thought to function in correct dynein assembly in cilia. Finally, we show that knock-down of polycystic kidney disease 2 (pkd2) specifically causes glomerular cysts and does not affect cilia motility, suggesting multiple mechanisms exist for cyst formation.
Project description:In this review, we will discuss several well-accepted signaling pathways toward calcium-mediated mechanisms of cystic expansion. The second messenger calcium ion has contributed to a vast diversity of signal transduction pathways. We will dissect calcium signaling as a possible mechanism that contributes to renal cyst formation. Because cytosolic calcium also regulates an array of signaling pathways, we will first discuss cilia-induced calcium fluxes, followed by Wnt signaling that has attributed to much-discussed planar cell polarity. We will then look at the relationship between cytosolic calcium and cAMP as one of the most important aspects of cyst progression. The signaling of cAMP on MAPK and mTOR will also be discussed. We infer that while cilia-induced calcium fluxes may be the initial signaling messenger for various cellular pathways, no single signaling mediator or pathway is implicated exclusively in the progression of the cystic expansion. This article is part of a Special Issue entitled: Polycystic Kidney Disease.
Project description:BACKGROUND:Cystic kidneys and vascular aneurysms are clinical manifestations seen in patients with polycystic kidney disease, a cilia-associated pathology (ciliopathy). Survivin overexpression is associated with cancer, but the clinical pathology associated with survivin downregulation or knockout has never been studied before. The present studies aim to examine whether and how cilia function (Pkd1 or Pkd2) and structure (Tg737) play a role in cystic kidney and aneurysm through survivin downregulation. METHODS AND RESULTS:Cysts and aneurysms from polycystic kidney disease patients, Pkd mouse, and zebrafish models are characterized by chromosome instability and low survivin expression. This triggers cytokinesis defects and formation of nuclear polyploidy or aneuploidy. In vivo conditional mouse and zebrafish models confirm that survivin gene deletion in the kidneys results in a cystic phenotype. As in hypertensive Pkd1, Pkd2, and Tg737 models, aneurysm formation can also be induced in vascular-specific normotensive survivin mice. Survivin knockout also contributes to abnormal oriented cell division in both kidney and vasculature. Furthermore, survivin expression and ciliary localization are regulated by flow-induced cilia activation through protein kinase C, Akt and nuclear factor-?B. Circumventing ciliary function by re-expressing survivin can rescue polycystic kidney disease phenotypes. CONCLUSIONS:For the first time, our studies offer a unifying mechanism that explains both renal and vascular phenotypes in polycystic kidney disease. Although primary cilia dysfunction accounts for aneurysm formation and hypertension, hypertension itself does not cause aneurysm. Furthermore, aneurysm formation and cyst formation share a common cellular and molecular pathway involving cilia function or structure, survivin expression, cytokinesis, cell ploidy, symmetrical cell division, and tissue architecture orientation.
Project description:Intraflagellar transport (IFT) complexes A and B build and maintain primary cilia. In the mouse, kidney-specific or hypomorphic mutant alleles of IFT complex B genes cause polycystic kidneys, but the influence of IFT complex A proteins on renal development is not well understood. In the present study, we found that HoxB7-Cre-driven deletion of the complex A gene Ift140 from collecting ducts disrupted, but did not completely prevent, cilia assembly. Mutant kidneys developed collecting duct cysts by postnatal day 5, with rapid cystic expansion and renal dysfunction by day 15 and little remaining parenchymal tissue by day 20. In contrast to many models of polycystic kidney disease, precystic Ift140-deleted collecting ducts showed normal centrosomal positioning and no misorientation of the mitotic spindle axis, suggesting that disruption of oriented cell division is not a prerequisite to cyst formation in these kidneys. Precystic collecting ducts had an increased mitotic index, suggesting that cell proliferation may drive cyst expansion even with normal orientation of the mitotic spindle. In addition, we observed significant increases in expression of canonical Wnt pathway genes and mediators of Hedgehog and tissue fibrosis in highly cystic, but not precystic, kidneys. Taken together, these studies indicate that loss of Ift140 causes pronounced renal cystic disease and suggest that abnormalities in several different pathways may influence cyst progression.
Project description:Coxiella burnetii, the causative agent of Q fever, is a zoonotic disease with potentially life-threatening complications in humans. Inhalation of low doses of Coxiella bacteria can result in infection of the host alveolar macrophage (AM). However, it is not known whether a subset of AMs within the heterogeneous population of macrophages in the infected lung is particularly susceptible to infection. We have found that lower doses of both phase I and phase II Nine Mile C. burnetii multiply and are less readily cleared from the lungs of mice compared to higher infectious doses. We have additionally identified AM resident within the lung prior to and shortly following infection, opposed to newly recruited monocytes entering the lung during infection, as being most susceptible to infection. These resident cells remain infected up to twelve days after the onset of infection, serving as a permissive niche for the maintenance of bacterial infection. A subset of infected resident AMs undergo a distinguishing phenotypic change during the progression of infection exhibiting an increase in surface integrin CD11b expression and continued expression of the surface integrin CD11c. The low rate of phase I and II Nine Mile C. burnetii growth in murine lungs may be a direct result of the limited size of the susceptible resident AM cell population.
Project description:Primary cilia project from the surface of most vertebrate cells and are thought to be sensory organelles. Defects in primary cilia lead to cystic kidney disease, although the ciliary mechanisms that promote and maintain normal renal function remain incompletely understood. In this work, we generated a floxed allele of the ciliary assembly gene Ift20. Deleting this gene specifically in kidney collecting duct cells prevents cilia formation and promotes rapid postnatal cystic expansion of the kidney. Dividing collecting duct cells in early stages of cyst formation fail to properly orient their mitotic spindles along the tubule, whereas nondividing cells improperly position their centrosomes. At later stages, cells lacking cilia have increased canonical Wnt signaling and increased rates of proliferation. Thus, IFT20 functions to couple extracellular events to cell proliferation and differentiation.
Project description:BACKGROUND:Neovascular age-related macular degeneration (nAMD) commonly causes vision loss from aberrant angiogenesis, termed choroidal neovascularization (CNV). Macrophages are heterogeneous cells that are necessary for experimental CNV, present in human CNV samples, and can display diverse functions, which are dependent upon both their origin and tissue microenvironment. Despite these associations, choroidal macrophage heterogeneity remains unexplored. METHODS:We performed multi-parameter flow cytometry on wildtype (WT) and Ccr2-/- mice after laser injury to identify macrophage subtypes, and determine which subsets originate from classical monocytes. To fate map tissue resident macrophages at steady state and after laser injury, we used the Cx3cr1CreER/+ ; Rosa26zsGFP/+ mouse model. We reanalyzed previously published single-cell RNA-seq of human choroid samples from healthy and nAMD patients to investigate human macrophage heterogeneity, disease association, and function. RESULTS:We identified 4 macrophage subsets in mice: microglia, MHCII+CD11c-, MHCII+CD11c+, and MHCII-. Microglia are tissue resident macrophages at steady state and unaffected by laser injury. At steady state, MHCII- macrophages are long lived, tissue resident macrophages, while MHCII+CD11c- and MHCII+CD11c+ macrophages are partially replenished from blood monocytes. After laser injury, MHCII+CD11c- macrophages are entirely derived from classical monocytes, MHCII- macrophages originate from classical monocytes (90%) and an expansion of tissue resident macrophages (10%), and MHCII+CD11c+ macrophages are derived from classical monocytes (70%), non-classical monocytes (10%), and an expansion of tissue resident macrophages (20%). Single-cell RNA-seq analysis of human choroid found 5 macrophage subsets: two MHCII+CD11C- and three MHCII+CD11C+ populations. One MHCII+CD11C+ subset was 78% derived from a patient with nAMD. Differential expression analysis identified up-regulation of pro-angiogenic gene expression in one MHCII+CD11C- and two MHCII+CD11C+ subsets, including the disease-associated cluster. The upregulated MHCII+CD11C- pro-angiogenic genes were unique compared to the increased MHCII+CD11C+ angiogenesis genes. CONCLUSIONS:Macrophage origin impacts heterogeneity at steady state and after laser injury in mice. Both mice and human patients demonstrate similar macrophage subtypes. Two discrete pro-angiogenic macrophage populations exist in the human choroid. Targeting specific, pro-angiogenic macrophage subsets is a potential novel therapeutic for nAMD.
Project description:Polycystic liver disease (PLD) is a member of the cholangiopathies, a group of liver diseases in which cholangiocytes, the epithelia lining of the biliary tree, are the target cells. PLDs are caused by mutations in genes involved in intracellular signaling pathways, cell cycle regulation, and ciliogenesis, among others. We previously showed that cystic cholangiocytes have abnormal cell cycle profiles and malfunctioning cilia. Because histone deacetylase 6 (HDAC6) plays an important role in both cell cycle regulation and ciliary disassembly, we examined the role of HDAC6 in hepatic cystogenesis. HDAC6 protein was increased sixfold in cystic liver tissue and in cultured cholangiocytes isolated from both PCK rats (an animal model of PLD) and humans with PLD. Furthermore, pharmacological inhibition of HDAC6 by Tubastatin-A, Tubacin, and ACY-1215 decreased proliferation of cystic cholangiocytes in a dose- and time-dependent manner, and inhibited cyst growth in three-dimensional cultures. Importantly, ACY-1215 administered to PCK rats diminished liver cyst development and fibrosis. In summary, we show that HDAC6 is overexpressed in cystic cholangiocytes both in vitro and in vivo, and its pharmacological inhibition reduces cholangiocyte proliferation and cyst growth. These data suggest that HDAC6 may represent a potential novel therapeutic target for cases of PLD.