We showed that renal proximal tubular cells (RPTC) can proliferate and migrate following plating and oxidant or mechanical injury in the absence of exogenous growth factors; however, the mechanisms of this response remain unclear. We examined whether epidermal growth factor receptor (EGFR) signaling is activated following plating and mechanical injury and mediates RPTC proliferation and migration. EGFR, Akt [a target of phosphoinositide-3-kinase (PI3K)], and ERK1/2 were activated after plating and mechanical injury, and their phosphorylation was further enhanced by addition of exogenous EGF. Inactivation of the EGFR with the selective inhibitor AG-1478 completely blocked phosphorylation of EGFR, Akt, and ERK1/2 and blocked cell proliferation and migration after plating and injury. Inhibition of PI3K with LY-294002 blocked Akt phosphorylation and proliferation, whereas U-0126 blocked ERK1/2 phosphorylation but had no effect on proliferation. Furthermore, p38 was phosphorylated following mechanical injury and the p38 inhibitor SB-203580 blocked p38 phosphorylation and cell migration. In contrast, neither PI3K nor ERK1/2 inhibition blocked cell migration. These results show that EGFR activation is required for RPTC proliferation and migration and that proliferation is mediated by PI3K, whereas migration is mediated by p38.

We previously have shown that the highly conserved eight-protein exocyst trafficking complex is required for ciliogenesis in kidney tubule cells. We hypothesized here that ciliogenic programs are conserved across organs and species. To determine whether renal primary ciliogenic programs are conserved in the eye, and to characterize the function and mechanisms by which the exocyst regulates eye development in zebrafish, we focused on , a central component of the exocyst complex, by analyzing both zebrafish mutants, and photoreceptor-specific Exoc5 knock-out mice. Two separate mutant zebrafish lines phenocopied morphants and, strikingly, exhibited a virtual absence of photoreceptors, along with abnormal retinal development and cell death. Because the zebrafish mutant was a global knockout, we also observed defects in several ciliated organs, including the brain (hydrocephalus), heart (cardiac edema), and kidney (disordered and shorter cilia). knockout increased phosphorylation of the regulatory protein Mob1, consistent with Hippo pathway activation. mutant zebrafish rescue with human EXOC5 mRNA completely reversed the mutant phenotype. We accomplished photoreceptor-specific knockout of Exoc5 with our fl/fl mouse line crossed with a rhodopsin-Cre driver line. In photoreceptor-specific knock-out mice, the photoreceptor outer segment structure was severely impaired at 4 weeks of age, although a full-field electroretinogram indicated a visual response was still present. However, by 6 weeks, visual responses were eliminated. In summary, we show that ciliogenesis programs are conserved in the kidneys and eyes of zebrafish and mice and that the exocyst is necessary for photoreceptor ciliogenesis and retinal development, most likely by trafficking cilia and outer-segment proteins.

Opening of voltage dependent anion channels (VDAC) by the erastin-like compound X1 and the multikinase inhibitor sorafenib promotes oxidative stress and mitochondrial dysfunction in hepatocarcinoma cells. Here, we hypothesized that X1 and sorafenib induce mitochondrial dysfunction by increasing ROS formation and activating c-Jun N-terminal kinases (JNKs), leading to translocation of activated JNK to mitochondria. Both X1 and sorafenib increased production of reactive oxygen species (ROS) and activated JNK. X1 and sorafenib caused a drop in mitochondrial membrane potential (ΔΨ), a readout of mitochondrial metabolism, after 60 min. Mitochondrial depolarization after X1 and sorafenib occurred in parallel with JNK activation, increased superoxide (O 2•− ) production, decreased basal and oligomycin sensitive respiration, and decreased maximal respiratory capacity. Increased production of O 2 •− after X1 or sorafenib was abrogated by JNK inhibition and antioxidants. S3QEL specific inhibitor of site IIIQ o, at Complex III prevented depolarization induced by X1. JNK inhibition by JNK inhibitors VIII and SP600125 also prevented mitochondrial depolarization. After X1, activated JNK translocated to mitochondria as assessed by proximity ligation assays.

BackgroundThe disintegrin and metalloenzyme ADAM17 participates in numerous inflammatory and proliferative diseases, and its pathophysiological role was implicated in kidney fibrosis, polycystic kidney disease and other chronic kidney diseases. At present, we have little understanding how the enzyme activity is regulated. In this study we wanted to characterize the role of α5β1 integrin in ADAM17 activity regulation during G protein-coupled receptor (GPCR) stimulation.Methodology/Principal FindingsWe showed previously that the profibrotic GPCR agonist serotonin (5-HT) induced kidney mesangial cell proliferation through ADAM17 activation and heparin-binding epidermal growth factor (HB-EGF) shedding. In the present studies we observed that in unstimulated mesangial cell lysates α5β1 integrin co-precipitated with ADAM17 and that 5-HT treatment of the cells induced dissociation of α5β1 integrin from ADAM17. Using fluorescence immunostaining and in situ proximity ligation assay, we identified the perinuclear region as the localization of the ADAM17/α5β1 integrin interaction. In cell-free assays, we showed that purified α5β1 integrin and β1 integrin dose-dependently bound to and inhibited activity of recombinant ADAM17. We provided evidence that the conformation of the integrin determines its ADAM17-binding ability. To study the effect of β1 integrin on ADAM17 sheddase activity, we employed alkaline phosphatase-tagged HB-EGF. Overexpression of β1 integrin lead to complete inhibition of 5-HT-induced HB-EGF shedding and silencing β1 integrin by siRNA significantly increased mesangial cells ADAM17 responsiveness to 5-HT.Conclusions/SignificanceOur data show for the first time that β1 integrin has an important physiological role in ADAM17 activity regulation. We suggest that regulating α5β1 integrin binding to ADAM17 could be an attractive therapeutic target in chronic kidney diseases.

Background: Bicuspid aortic valve (BAV) disease is a congenital defect that affects 0.5% to 1.2% of the population and is associated with comorbidities including ascending aortic dilation and calcific aortic valve stenosis. To date, although a few causal genes have been identified, the genetic basis for the vast majority of BAV cases remains unknown, likely pointing to complex genetic heterogeneity underlying this phenotype. Identifying genetic pathways versus individual gene variants may provide an avenue for uncovering additional BAV causes and consequent comorbidities. Methods: We performed genome-wide association Discovery and Replication Studies using cohorts of 2131 patients with BAV and 2728 control patients, respectively, which identified primary cilia genes as associated with the BAV phenotype. Genome-wide association study hits were prioritized based on P value and validated through in vivo loss of function and rescue experiments, 3-dimensional immunohistochemistry, histology, and morphometric analyses during aortic valve morphogenesis and in aged animals in multiple species. Consequences of these genetic perturbations on cilia-dependent pathways were analyzed by Western and immunohistochemistry analyses, and assessment of aortic valve and cardiac function were determined by echocardiography. Results: Genome-wide association study hits revealed an association between BAV and genetic variation in human primary cilia. The most associated single-nucleotide polymorphisms were identified in or near genes that are important in regulating ciliogenesis through the exocyst, a shuttling complex that chaperones cilia cargo to the membrane. Genetic dismantling of the exocyst resulted in impaired ciliogenesis, disrupted ciliogenic signaling and a spectrum of cardiac defects in zebrafish, and aortic valve defects including BAV, valvular stenosis, and valvular calcification in murine models. Conclusions: These data support the exocyst as required for normal ciliogenesis during aortic valve morphogenesis and implicate disruption of ciliogenesis and its downstream pathways as contributory to BAV and associated comorbidities in humans.