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Autosomal Dominant Polycystic Kidney Disease (ADPKD) is caused by mutation of PKD1 or PKD2 , which encode polycystin 1 and 2, respectively. The polycystins localize to primary cilia and the functional loss of the polycystin complex leads to the formation and progressive growth of fluid-filled cysts in the kidney. The pathogenesis of ADPKD is complex and molecular mechanisms connecting ciliary dysfunction to renal cystogenesis are unclear. Primary cilia mediate Hedgehog signaling, which modulates cell proliferation and differentiation in a tissue-dependent manner. Previously, we showed that Hedgehog signaling was increased in cystic kidneys of several PKD mouse models and that Hedgehog inhibition prevented cyst formation in embryonic PKD mouse kidneys treated with cAMP. Here, we show that in human ADPKD tissue, Hedgehog target and activator, Glioma 1, was elevated and localized to cyst-lining epithelial cells and to interstitial cells, suggesting increased autocrine and paracrine Hedgehog signaling in ADPKD, respectively. Further, Hedgehog inhibitors reduced basal and cAMP-induced proliferation of ADPKD cells and cyst formation in vitro . These data suggest that Hedgehog signaling is increased in human ADPKD and that suppression of Hedgehog signaling can counter cellular processes that promote cyst growth in vitro .

Primary cilia are small, antenna-like organelles that detect and transduce chemical and mechanical cues in the extracellular environment, regulating cell behavior and, in turn, tissue development and homeostasis. Primary cilia are assembled via intraflagellar transport (IFT), which traffics protein cargo bidirectionally along a microtubular axoneme. Ranging from 1 to 10 μm long, these organelles typically reach a characteristic length dependent on cell type, likely for optimum fulfillment of their specific roles. The importance of an optimal cilia length is underscored by the findings that perturbation of cilia length can be observed in a number of cilia-related diseases. Thus, elucidating mechanisms of cilia length regulation is important for understanding the pathobiology of ciliary diseases. Since cilia assembly/disassembly regulate cilia length, we review the roles of IFT in processes that affect cilia assembly/disassembly, including ciliary transport of structural and membrane proteins, ectocytosis, and tubulin posttranslational modification. Additionally, since the environment of a cell influences cilia length, we also review the various stimuli encountered by renal epithelia in healthy and diseased states that alter cilia length and IFT.

Polycystic kidney disease (PKD) is a genetic disorder characterized by fluid-filled cysts in the kidney and liver that ultimately leads to end-stage renal disease. Currently there is no globally approved therapy for PKD. The Notch signaling pathway regulates cellular processes such as proliferation and de-differentiation, which are cellular hallmarks of PKD. Thus we hypothesized that the Notch pathway plays a critical role in PKD. Evaluation of protein expression of Notch signaling components in kidneys of Autosomal Recessive PKD (ARPKD) and Autosomal Dominant PKD (ADPKD) mouse models and of ADPKD patients revealed that Notch pathway members, particularly Notch3, were consistently upregulated or activated in cyst-lining epithelial cells. Notch3 expression correlated with rapidly growing cysts and co-localized with the proliferation marker, PCNA. Importantly, Notch inhibition significantly decreased forskolin-induced Notch3 activation and proliferation of primary human ADPKD cells, and significantly reduced cyst formation and growth of human ADPKD cells cultured in collagen gels. Thus our data indicate that Notch3 is aberrantly activated and facilitates epithelial cell proliferation in PKD, and that inhibition of Notch signaling may prevent cyst formation and growth.

Anti-retroviral therapy (ART) has decreased human immunodeficiency virus (HIV)-1-associated morbidity. However, despite ART, immune cells remain latently infected, leading to chronic inflammation and HIV-1-associated comorbidities. New strategies are needed to target viral proteins and inflammation. We found activation of Notch3 in renal cells of the HIV-1 transgenic mouse model (HIV-Tg26) and in patients with HIV-associated nephropathy. We hypothesized that targeting NOTCH3 activation constitutes an effective therapy for HIV-related chronic kidney disease. We generated HIV-Tg26 mice with Notch3 knocked out (Tg-N3KO). Compared to HIV-Tg26 mice at 3 months, Tg-N3KO mice showed a marked reduction in renal injury, skin lesions and mortality rate. They also showed reduced renal infiltrating cells and significantly reduced expression of HIV genes. Moreover, Notch3 activated the HIV long terminal repeat promoter, and induction of HIV-1 increased Notch3 activation, indicating a feedback mechanism. Further, bone marrow-derived macrophages from HIV-Tg26 mice showed activation of Notch3, indicating systemic effects. Consistent with that observation, systemic levels of TNF and MCP-1 were reduced in Tg-N3KO compared to HIV-Tg26 mice. Thus, Notch3 deletion/inhibition has a dual-therapeutic effect in HIV-related chronic kidney disease, which might extend to other HIV-related pathologies.

Primary cilia are organelles extended from virtually all cells and are required for the proper regulation of a number of canonical developmental pathways. The role in cortical development of proteins important for ciliary form and function is a relatively understudied area. Here we have taken a genetic approach to define the role in forebrain development of three intraflagellar transport proteins known to be important for primary cilia function. We have genetically ablated Kif3a, Ift88, and Ttc21b in a series of specific spatiotemporal domains. The resulting phenotypes allow us to draw several conclusions. First, we conclude that the Ttc21b cortical phenotype is not due to the activity of Ttc21b within the brain itself. Secondly, some of the most striking phenotypes are from ablations in the neural crest cells and the adjacent surface ectoderm indicating that cilia transduce critical tissue-tissue interactions in the developing embryonic head. Finally, we note striking differences in phenotypes from ablations only one embryonic day apart, indicating very discrete spatiotemporal requirements for these three genes in cortical development.

Despite the recent launch of tolvaptan, the search for safer polycystic kidney disease (PKD) drugs continues. Ciclopirox (CPX) or its olamine salt (CPX-O) is contained in a number of commercially available antifungal agents. CPX is also reported to possess anticancer activity. Several mechanisms of action have been proposed, including chelation of iron and inhibition of iron-dependent enzymes. Here, we show that CPX-O inhibited in vitro cystogenesis of primary human PKD cyst-lining epithelial cells cultured in a 3D collagen matrix. To assess the in vivo role of CPX-O, we treated PKD mice with CPX-O. CPX-O reduced the kidney-to-body weight ratios of PKD mice. The CPX-O treatment was also associated with decreased cell proliferation, decreased cystic area, and improved renal function. Ferritin levels were markedly elevated in cystic kidneys of PKD mice, and CPX-O treatment reduced renal ferritin levels. The reduction in ferritin was associated with increased ferritinophagy marker nuclear receptor coactivator 4, which reversed upon CPX-O treatment in PKD mice. Interestingly, these effects on ferritin appeared independent of iron. These data suggest that CPX-O can induce ferritin degradation via ferritinophagy, which is associated with decreased cyst growth progression in PKD mice. Most importantly these data indicate that CPX-O has the potential to treat autosomal dominant PKD.

Development of the embryonic palate requires that the palatal shelves (PS), which extend from maxillary processes, to grow bilaterally and vertically alongside the tongue. This growth continues until embryonic day (E) 13.5, after which the PS elevate above the tongue and adhere, completing the process by E14.5. Current models indicate that this elevation process involves a complex vertical-to-horizontal PS reorientation. While earlier studies have implied that reorientation occurs rapidly, the precise timing has not been resolved. Time-restricted pregnancies with a 1-h resolution showed that in 97% of C57BL/6J embryos, the PS were unelevated at E14.0. However, 6 h later, at E14.25, the PS had completed elevation in 80% of embryos, indicating that the PS elevate in a rapid and constrained timeframe. Interestingly, all E14.25 embryos with unelevated PS (20%) were female, suggesting sex differences in C57BL/6J PS elevation. In FVB/NJ embryos, the elevation window started earlier (E13.875-E14.25), and without any sex differences. An intermediate stage with unilateral PS elevation was frequently observed. Magnetic resonance imaging (MRI) of various stages showed that PS elevation began with posterior bilateral bulges, which then progressed laterally and anteriorly over time. During elevation, we observed increased cell proliferation in the PS lingual region. Within the bulge, cell orientation was tilted towards the tongue, and actomyosin activity was increased, which together may participate in horizontal projection of the bulge. Thus, our data reveal novel insights into rapid dynamic changes during PS elevation, and lay the foundation for future studies of normal and abnormal palatogenesis.

We conducted a genetic analysis of the developing temporo-mandibular or temporomandi-bular joint (TMJ), a highly specialized synovial joint that permits movement and function of the mammalian jaw. First, we used laser capture microdissection to perform a genome-wide expression analysis of each of its developing components. The expression patterns of genes identified in this screen were examined in the TMJ and compared with those of other synovial joints, including the shoulder and the hip joints. Striking differences were noted, indicating that the TMJ forms via a distinct molecular program. Several components of the hedgehog (Hh) signaling path-way are among the genes identified in the screen, including Gli2, which is expressed specifically in the condyle and in the disk of the developing TMJ. We found that mice deficient in Gli2 display aberrant TMJ development such that the condyle loses its growth-plate-like cellular organization and no disk is formed. In addition, we used a conditional strategy to remove Smo, a positive effector of the Hh signaling pathway, from chondrocyte progenitors. This cell autonomous loss of Hh signaling allows for disk formation, but the resulting structure fails to separate from the condyle. Thus, these experiments establish that Hh signaling acts at two distinct steps in disk morphogenesis, condyle initiation, and disk-condyle separation and provide a molecular framework for future studies of the TMJ.

Ciliopathies are genetic syndromes that link skeletal dysplasias to the dysfunction of primary cilia. Primary cilia are sensory organelles synthesized by intraflagellar transport (IFT)—A and B complexes, which traffic protein cargo along a microtubular core. We have reported that the deletion of the IFT-A gene, Thm2, together with a null allele of its paralog, Thm1, causes a small skeleton with a small mandible or micrognathia in juvenile mice. Using micro-computed tomography, here we quantify the craniofacial defects of Thm2−/−; Thm1aln/+ triple allele mutant mice. At postnatal day 14, triple allele mutant mice exhibited micrognathia, midface hypoplasia, and a decreased facial angle due to shortened upper jaw length, premaxilla, and nasal bones, reflecting altered development of facial anterior-posterior elements. Mutant mice also showed increased palatal width, while other aspects of the facial transverse, as well as vertical dimensions, remained intact. As such, other ciliopathy-related craniofacial defects, such as cleft lip and/or palate, hypo-/hypertelorism, broad nasal bridge, craniosynostosis, and facial asymmetry, were not observed. Calvarial-derived osteoblasts of triple allele mutant mice showed reduced bone formation in vitro that was ameliorated by Hedgehog agonist, SAG. Together, these data indicate that Thm2 and Thm1 genetically interact to regulate bone formation and sculpting of the postnatal face. The triple allele mutant mice present a novel model to study craniofacial bone development.

Primary cilia extend from the plasma membrane of most vertebrate cells and mediate signaling pathways. Ciliary dysfunction underlies ciliopathies, which are genetic syndromes that manifest multiple clinical features, including renal cystic disease and obesity. THM1 (also termed TTC21B or IFT139) encodes a component of the intraflagellar transport-A complex and mutations in THM1 have been identified in 5% of individuals with ciliopathies. Consistent with this, deletion of murine Thm1 during late embryonic development results in cystic kidney disease. Here we report that deletion of murine Thm1 during adulthood results in obesity, diabetes, hypertension and fatty liver disease, with gender differences in susceptibility to weight gain and metabolic dysfunction. Pair-feeding of Thm1 conditional knock-out mice relative to control littermates prevented the obesity and related disorders, indicating that hyperphagia caused the obese phenotype. Thm1 ablation resulted in increased localization of adenylyl cyclase III in primary cilia that were shortened with bulbous distal tips on neurons of the hypothalamic arcuate nucleus, an integrative center for signals that regulate feeding and activity. In pre-obese Thm1 conditional knock-out mice, expression of anorexogenic pro-opiomelanocortin was decreased by 50% in the arcuate nucleus, which likely caused the hyperphagia. Fasting of Thm1 conditional knock-out mice did not alter pro-opiomelanocortin nor orexogenic agouti-related peptide expression, suggesting impaired sensing of changes in peripheral signals. Together, these data indicate that the Thm1-mutant ciliary defect diminishes sensitivity to feeding signals, which alters appetite regulation and leads to hyperphagia, obesity and metabolic disease.