Julie A. Jonassen

Deletion of IFT20 in the mouse kidney causes misorientation of the mitotic spindle and cystic kidney disease

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.

The Golgin GMAP210/TRIP11 anchors IFT20 to the Golgi complex

Eukaryotic cells often use proteins localized to the ciliary membrane to monitor the extracellular environment. The mechanism by which proteins are sorted, specifically to this subdomain of the plasma membrane, is almost completely unknown. Previously, we showed that the IFT20 subunit of the intraflagellar transport particle is localized to the Golgi complex, in addition to the cilium and centrosome, and hypothesized that the Golgi pool of IFT20 plays a role in sorting proteins to the ciliary membrane. Here, we show that IFT20 is anchored to the Golgi complex by the golgin protein GMAP210/Trip11. Mice lacking GMAP210 die at birth with a pleiotropic phenotype that includes growth restriction, ventricular septal defects of the heart, omphalocele, and lung hypoplasia. Cells lacking GMAP210 have normal Golgi structure, but IFT20 is no longer localized to this organelle. GMAP210 is not absolutely required for ciliary assembly, but cilia on GMAP210 mutant cells are shorter than normal and have reduced amounts of the membrane protein polycystin-2 localized to them. This work suggests that GMAP210 and IFT20 function together at the Golgi in the sorting or transport of proteins destined for the ciliary membrane.

IFT25 links the signal-dependent movement of Hedgehog components to intraflagellar transport.

The intraflagellar transport (IFT) system is required for building primary cilia, sensory organelles that cells use to respond to their environment. IFT particles are composed of about 20 proteins, and these proteins are highly conserved across ciliated species. IFT25, however, is absent from some ciliated organisms, suggesting that it may have a unique role distinct from ciliogenesis. Here, we generate an Ift25 null mouse and show that IFT25 is not required for ciliary assembly but is required for proper Hedgehog signaling, which in mammals occurs within cilia. Mutant mice die at birth with multiple phenotypes, indicative of Hedgehog signaling dysfunction. Cilia lacking IFT25 have defects in the signal-dependent transport of multiple Hedgehog components including Patched-1, Smoothened, and Gli2, and fail to activate the pathway upon stimulation. Thus, IFT function is not restricted to building cilia where signaling occurs, but also plays a separable role in signal transduction events.

IFT27 Links the BBSome to IFT for Maintenance of the Ciliary Signaling Compartment

Vertebrate hedgehog signaling is coordinated by the differential localization of the receptors patched-1 and Smoothened in the primary cilium. Cilia assembly is mediated by intraflagellar transport (IFT), and cilia defects disrupt hedgehog signaling, causing many structural birth defects. We generated Ift25 and Ift27 knockout mice and show that they have structural birth defects indicative of hedgehog signaling dysfunction. Surprisingly, ciliary assembly is not affected, but abnormal hedgehog signaling is observed in conjunction with ciliary accumulation of patched-1 and Smoothened. Similarly, Smoothened accumulates in cilia on cells mutated for BBSome components or the BBS binding protein/regulator Lztfl1. Interestingly, the BBSome and Lztfl1 accumulate to high levels in Ift27 mutant cilia. Because Lztfl1 mutant cells accumulate BBSome but not IFT27, it is likely that Lztfl1 functions downstream of IFT27 to couple the BBSome to the IFT particle for coordinated removal of patched-1 and Smoothened from cilia during hedgehog signaling.

Disruption of IFT Complex A Causes Cystic Kidneys without Mitotic Spindle Misorientation

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.

Oxalate toxicity in LLC-PK1 cells, a line of renal epithelial cells

PURPOSE The present studies assessed the possibility that high concentrations of oxalate may be toxic to renal epithelial cells. MATERIALS AND METHODS Subconfluent cultures of LLC-PK1 cells were exposed to oxalate, and the effects on cell morphology, membrane permeability to vital dyes, DNA integrity and cell density were assessed. RESULTS Oxalate exposure produced time- and concentration-dependent changes in the light microscopic appearance of LLC-PK1 cells with higher concentrations ( > 140 microM.) inducing marked cytosolic vacuolization and nuclear pyknosis. Exposure to oxalate also increased membrane permeability to vital dyes, promoted DNA fragmentation and, at high concentrations (350 microM. free oxalate), induced a net loss of LLC-PK1 cells. CONCLUSIONS Since high concentrations of oxalate can be toxic to renal epithelial cells, hyperoxaluria may contribute to several forms of renal disease including both calcium stone disease and end-stage renal disease.

Oxalate toxicity in renal cells

Exposure to oxalate, a constituent of the most common form of kidney stones, generates toxic responses in renal epithelial cells, including altered membrane surface properties and cellular lipids, changes in gene expression, disruption of mitochondrial function, formation of reactive oxygen species and decreased cell viability. Oxalate exposure activates phospholipase A2 (PLA2), which increases two lipid signaling molecules, arachidonic acid and lysophosphatidylcholine (Lyso-PC). PLA2 inhibition blocks, whereas exogenous Lyso-PC or arachidonic acid reproduce many of the effects of oxalate on mitochondrial function, gene expression and cell viability, suggesting that PLA2 activation plays a role in mediating oxalate toxicity. Oxalate exposure also elicits potentially adaptive or protective changes that increase expression of proteins that may prevent crystal formation or attachment. Additional adaptive responses may facilitate removal and replacement of dead or damaged cells. The presence of different inflammatory cells and molecules in the kidneys of rats with hyperoxaluria and in stone patients suggests that inflammatory responses play roles in stone disease. Renal epithelial cells can synthesize a variety of cytokines, chemoattractants and other molecules with the potential to interface with inflammatory cells; moreover, oxalate exposure increases the synthesis of these molecules. The present studies demonstrate that oxalate exposure upregulates cyclooxygenase-2, which catalyzes the rate-limiting step in the synthesis of prostanoids, compounds derived from arachidonic acid that can modify crystal binding and may also influence inflammation. In addition, renal cell oxalate exposure promotes rapid degradation of IκBα, an endogenous inhibitor of the NF-κB transcription factor. A similar response is observed following renal cell exposure to lipopolysaccharide (LPS), a bacterial cell wall component that activates toll-like receptor 4 (TLR4). While TLRs are primarily associated with immune cells, they are also found on many other cell types, including renal epithelial cells, suggesting that TLR signaling could directly impact renal function. Prior exposure of renal epithelial cells to oxalate in vitro produces endotoxin tolerance, i.e. a loss of responsiveness to LPS and conversely, prior exposure to LPS elicits a similar heterologous desensitization to oxalate. Renal cell desensitization to oxalate stimulation may have profound effects on the outcome of renal stone disease by impairing protective responses.

Oxalate-induced redistribution of phosphatidylserine in renal epithelial cells: implications for kidney stone disease.

Aims: The present studies assessed the possibility that exposure to oxalate leads to alterations in membrane structure that promote crystal binding to renal epithelial cells. Specifically, we determined whether oxalate exposure produces a redistribution of membrane phosphatidylserine (PS) and an increase in the binding of 14C-oxalate crystals to renal epithelial cells. Methods: PS distribution was monitored in MDCK cells and in phospholipid-containing vesicles using NBD-PS, a fluorescent derivative of PS. Superfical PS was also detected by monitoring the binding of annexin V to MDCK cells. Results: Oxalate exposure rapidly increased the abundance of superficial NBD-PS and increased the binding of annexin V to MDCK cells. Oxalate exposure also increased PS at the surface of phospholipid vesicles, suggesting that oxalate may interact directly with PS. The oxalate concentrations that increased superficial PS also increased binding of 14C-oxalate crystals to MDCK cells, and the increased crystal binding was blocked by annexin V. Conclusions: These findings provide direct evidence that oxalate exposure promotes both a redistribution of PS and an increase in crystal binding in renal epithelial cells and support the notion that oxalate toxicity may contribute to the development of stone disease by altering the properties of the renal epithelial cell membrane.

Sexual health innovations in undergraduate medical education

Recent national and global initiatives have drawn attention to the importance of sexual health to individuals’ well-being. These initiatives advocate enhancement of efforts to address this under-represented topic in health professions curricula. University of Massachusetts Medical School (UMMS) has undertaken a comprehensive effort to develop an integrated curriculum in sexual health. The UMMS project draws upon the expertise of a multidisciplinary faculty of clinicians, basic scientists, a medical ethicist, and educators. This article describes the projects genesis and development at UMMS, and reports on three innovations in sexual health education implemented as part of this endeavor.

Identification of physician and patient attributes that influence the likelihood of screening for intimate partner violence

Purpose. Effective assessment of intimate partner violence (IPV) demands that everyone at risk be screened. To identify potential barriers, paper-and-pencil case scenarios identified possible practitioner and patient attributes that influence IPV screening. Method. First-year residents responded to one of four short written scenarios describing a divorced female patient with nonlocalized abdominal pain; variables were patient’s age and abdominal bruising. Residents rated their likelihood of screening for IPV and seven other screening tasks and self-assessed their competence in performing each task. Regression analyses assessed the influence of resident and patient characteristics on screening likelihood. Results. Patient bruising, younger patient age, and resident self-assessed competence best predicted IPV screening. Men were less likely than women to screen for IPV. Conclusions. Although most physicians receive training on IPV in medical school, barriers to IPV screening still exist. Identifying obstacles to IPV risk-assessment is an essential prerequisite for improving educational programs that promote routine IPV screening.