Mks6 mutations reveal tissue- and cell type-specific roles for the cilia transition zone.

The transition zone (TZ) is a domain at the base of the cilium that is involved in maintaining ciliary compartment-specific sensory and signaling activity by regulating cilia protein composition. Mutations in TZ proteins result in cilia dysfunction, often causing pleiotropic effects observed in a group of human diseases classified as ciliopathies. The purpose of this study is to describe the importance of the TZ component Meckel-Grüber syndrome 6 ( Mks6) in several organ systems and tissues regarding ciliogenesis and cilia maintenance using congenital and conditional mutant mouse models. Similar to MKS, congenital loss of Mks6 is embryonic lethal, displaying cilia loss and altered cytoskeletal microtubule modifications but only in specific cell types. Conditional Mks6 mutants have a variable cystic kidney phenotype along with severe retinal degeneration with mislocalization of phototransduction cascade proteins. However, other phenotypes, such as anosmia and obesity, which are typically associated with cilia and TZ dysfunction, were not evident. These data indicate that despite Mks6 being a core TZ component, it has tissue- or cell type-specific functions important for cilia formation and cilia sensory and signaling activities. Lewis, W. R., Bales, K. L., Revell, D. Z., Croyle, M. J., Engle, S. E., Song, C. J., Malarkey, E. B., Uytingco, C. R., Shan, D., Antonellis, P. J., Nagy, T. R., Kesterson, R. A., Mrug, M. M., Martens, J. R., Berbari, N. F., Gross, A. K., Yoder, B. K. Mks6 mutations reveal tissue- and cell type-specific roles for the cilia transition zone.

Tissue-Resident Macrophages Promote Renal Cystic Disease.

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.

Heterozygous Pkhd1C642* mice develop cystic liver disease and proximal tubule ectasia that mimics radiographic signs of medullary sponge kidney.

Heterozygosity for human polycystic kidney and hepatic disease 1 ( PKHD1) mutations was recently associated with cystic liver disease and radiographic findings resembling medullary sponge kidney (MSK). However, the relevance of these associations has been tempered by a lack of cystic liver or renal disease in heterozygous mice carrying Pkhd1 gene trap or exon deletions. To determine whether heterozygosity for a smaller Pkhd1 defect can trigger cystic renal disease in mice, we generated and characterized mice with the predicted truncating Pkhd1C642* mutation in a region corresponding to the middle of exon 20 cluster of five truncating human mutations (between PKHD1G617fs and PKHD1G644*). Mouse heterozygotes or homozygotes for the Pkhd1C642* mutation did not have noticeable liver or renal abnormalities on magnetic resonance images during their first weeks of life. However, when aged to ~1.5 yr, the Pkhd1C642* heterozygotes developed prominent cystic liver changes; tissue analyses revealed biliary cysts and increased number of bile ducts without signs of congenital hepatic fibrosis-like portal field inflammation and fibrosis that was seen in Pkhd1C642* homozygotes. Interestingly, aged female Pkhd1C642* heterozygotes, as well as homozygotes, developed radiographic changes resembling MSK. However, these changes correspond to proximal tubule ectasia, not an MSK-associated collecting duct ectasia. In summary, by demonstrating that cystic liver and kidney abnormalities are triggered by heterozygosity for the Pkhd1C642* mutation, we provide important validation for relevant human association studies. Together, these investigations indicate that PKHD1 mutation heterozygosity (predicted frequency 1 in 70 individuals) is an important underlying cause of cystic liver disorders and MSK-like manifestations in a human population.

Interferon Regulatory Factor-5 in Resident Macrophage Promotes Polycystic Kidney Disease.

BACKGROUND: Autosomal dominant polycystic kidney disease is caused by genetic mutations in PKD1 or PKD2. Macrophages and their associated inflammatory cytokines promote cyst progression; however, transcription factors within macrophages that control cytokine production and cystic disease are unknown. METHODS: In these studies, we used conditional Pkd1 mice to test the hypothesis that macrophage-localized interferon regulatory factor-5 (IRF5), a transcription factor associated with production of cyst-promoting cytokines (TNFα, IL-6), is required for accelerated cyst progression in a unilateral nephrectomy (1K) model. Analyses of quantitative real-time PCR (qRT-PCR) and flow-cytometry data 3 weeks post nephrectomy, a time point before the onset of severe cystogenesis, indicate an accumulation of inflammatory infiltrating and resident macrophages in 1K Pkd1 mice compared with controls. qRT-PCR data from FACS cells at this time demonstrate that macrophages from 1K Pkd1 mice have increased expression of Irf5 compared with controls. To determine the importance of macrophage-localized Irf5 in cyst progression, we injected scrambled or IRF5 antisense oligonucleotide (ASO) in 1K Pkd1 mice and analyzed the effect on macrophage numbers, cytokine production, and renal cystogenesis 6 weeks post nephrectomy. RESULTS: Analyses of qRT-PCR and IRF5 ASO treatment significantly reduced macrophage numbers, Irf5 expression in resident-but not infiltrating-macrophages, and the severity of cystic disease. In addition, IRF5 ASO treatment in 1K Pkd1 mice reduced Il6 expression in resident macrophages, which was correlated with reduced STAT3 phosphorylation and downstream p-STAT3 target gene expression. CONCLUSIONS: These data suggest that Irf5 promotes inflammatory cytokine production in resident macrophages resulting in accelerated cystogenesis.

Intravital visualization of the primary cilium, tubule flow, and innate immune cells in the kidney utilizing an abdominal window imaging approach.

The renal primary cilium is a small microtubule-based appendage thought to have mechano/chemosensory roles detecting changes in the fluid passing through the nephron. Mutations affecting cilium structure or function of ciliary-localized proteins result in a spectrum of diseases termed ciliopathies, with prevalent phenotypes such as the formation of renal cysts and fibrosis. While many studies have been conducted using fixed kidney sections or live imaging of cells in culture to investigate the cilium, examination in the context of a living murine kidney remains to be conducted. Previously, our lab generated the SSTR3GFP mouse to study cilium dynamics in vivo and found novel cilium behaviors that occurred following alteration of heart rate, blood pressure, and tubule flow. In this manuscript, we utilize multiple transgenic mouse models and an abdominal window imaging approach to observe primary cilia and tubule flow dynamics, immune cell movement, and renal Ca2+ signaling as it occurs in real time within a live mouse kidney. We present this window method as an approach that can be used in combination with various fluorescently labeled transgenic mice to investigate renal physiology, pathology, and function in vivo in longitudinal studies for as long as 5weeks.