Fenoldopam Sensitizes Primary Cilia-Mediated Mechanosensing to Promote Osteogenic Intercellular Signaling and Whole Bone Adaptation

Bone cells actively respond to mechanical stimuli to direct bone formation, yet there is no current treatment strategy for conditions of low bone mass and osteoporosis designed to target the inherent mechanosensitivity of bone. Our group has previously identified the primary cilium as a critical mechanosensor within bone, and that pharmacologically targeting the primary cilium with fenoldopam can enhance osteocyte mechanosensitivity. Here, we demonstrate that potentiating osteocyte mechanosensing with fenoldopam in vitro promotes pro-osteogenic paracrine signaling to osteoblasts. Conversely, impairing primary cilia formation and the function of key ciliary mechanotransduction proteins attenuates this intercellular signaling cascade. We then utilize an in vivo model of load-induced bone formation to demonstrate that fenoldopam treatment sensitizes bones of both healthy and osteoporotic mice to mechanical stimulation. Furthermore, we show minimal adverse effects of this treatment and demonstrate that prolonged treatment biases trabecular bone adaptation. This work is the first to examine the efficacy of targeting primary cilia-mediated mechanosensing to enhance bone formation in osteoporotic animals. © 2022 American Society for Bone and Mineral Research (ASBMR).     

Polycystic kidney disease and the renal cilium

Polycystic kidney disease (PKD) is a common genetic condition characterized by the formation of fluid-filled cysts in the kidney. Mutations affecting several genes are known to cause PKD and the protein products of most of these genes localize to an organelle called the renal cilium. Renal cilia are non-motile, microtubule-based projections located on the apical surface of the epithelial cells that form the tubules and ducts of the kidney. With the exception of intercalated cells, each epithelial cell bears a single non-motile cilium that projects into the luminal space where it is thought to act as a flow sensor. The detection of fluid flow through the kidney by the renal cilium is hypothesized to regulate a number of pathways responsible for the maintenance of normal epithelial phenotype. Defects of the renal cilium lead to cyst formation, caused primarily by the dedifferentiation and over-proliferation of epithelial cells. Here we discuss the role of renal cilia and the mechanisms by which defects of this organelle are thought to lead to PKD.     

Formation of primary cilia in the renal epithelium is regulated by the von Hippel-Lindau tumor suppressor protein

Growing evidence points to defects in the primary cilium as a critical mechanism underlying renal cyst development. Inactivation of the VHL gene is responsible for the autosomal dominant condition von Hippel-Lindau (VHL) disease and is implicated in most sporadic clear cell renal carcinomas. Manifestations of VHL disease include cysts in several organs, particularly in the kidney. Here it is shown that VHL inactivation is associated with abrogation of the primary cilium in renal cysts of patients with VHL disease and in VHL-defective cell lines. Complementation of VHL-defective clear cell renal carcinoma cell lines with wild-type VHL restored primary cilia. Moreover, it is shown that the effects of VHL on the primary cilium are mediated substantially via hypoxia-inducible factor. The effect of VHL status on the primary cilium provides a potential mechanism for renal cyst development in VHL disease and may help in the understanding of how VHL acts as a tumor suppressor.     

The canonical Wnt signaling pathway is not involved in renal cyst development in the kidneys of inv mutant mice

Recent studies have identified several genes whose defects cause hereditary renal cystic diseases with most of the gene products located in the primary cilia. It has been suggested that primary cilia are involved in signaling pathways, defects of which result in abnormal cell proliferation and randomization of oriented cell division in the kidney leading to cyst formation. Mice with a mutation in the inv gene are a model for human nephronophthisis type 2 and develop multiple renal cysts. Inv protein (also called inversin) is located in the base of primary cilia and acts as a switch from canonical to non-canonical Wnt signaling. Here, we studied the orientation of cell division and proliferation in the kidneys of inv mutant mice, as its loss is thought to maintain activation of the canonical Wnt signaling. To establish if canonical signaling was involved in this process, we mated inv mutant with BATlacZ mice to measure canonical Wnt activity. Based on these reporter mice, nuclear localization and phosphorylation of β-catenin, and responsiveness to Wnt ligands in inv mutant cells, we found that random oriented cell division is an initial event for renal tubule expansion and precedes cell proliferation. Thus, our results do not support the hypothesis that canonical Wnt signaling causes renal cyst development in these mice.     

Loss of primary cilia upregulates renal hypertrophic signaling and promotes cystogenesis

Primary cilia dysfunction alters renal tubular cell proliferation and differentiation and associates with accelerated cyst formation in polycystic kidney disease. However, the mechanism leading from primary ciliary dysfunction to renal cyst formation is unknown. We hypothesize that primary cilia prevent renal cyst formation by suppressing pathologic tubular cell hypertrophy and proliferation. Unilateral nephrectomy initiates tubular cell hypertrophy and proliferation in the contralateral kidney and provides a tool to examine primary cilia regulation of renal hypertrophy. Conditional knockout of the primary cilia ift88 gene leads to delayed, adult-onset renal cystic disease, which provides a window of opportunity to conduct unilateral nephrectomy and examine downstream kinetics of renal hypertrophy and cyst formation. In wild-type animals, unilateral nephrectomy activated the mTOR pathway and produced appropriate structural and functional hypertrophy without renal cyst formation. However, in ift88 conditional knockout animals, unilateral nephrectomy triggered increased renal hypertrophy and accelerated renal cyst formation, leading to renal dysfunction. mTOR signaling also increased compared with wild-type animals, suggesting a mechanistic cascade starting with primary ciliary dysfunction, leading to excessive mTOR signaling and renal hypertrophic signaling and culminating in cyst formation. These data suggest that events initiating hypertrophic signaling, such as structural or functional loss of renal mass, may accelerate progression of adult polycystic kidney disease toward end-stage renal disease.     

Autosomal dominant polycystic kidney disease: recent advances in pathogenesis and potential therapies

Autosomal dominant polycystic kidney disease (ADPKD) is the most common progressive hereditary kidney disease. In 85–90 % of cases, ADPKD results from a mutation in the PKD1 gene, and the other 10–15 % of the cases are accounted for by mutations in PKD2. PKD1 and PKD2 encode polycystin-1 and polycystin-2. Polycystin-1 may be a receptor that controls the channel activity of polycystin-2 as part of the polycystin signaling complex. ADPKD is characterized by the progressive development of fluid-filled cysts derived from renal tubular epithelial cells that gradually compress the parenchyma and compromise renal function. In recent years, considerable interest has developed in the primary cilia as a site of the proteins that are involved in renal cystogenesis. The pathological processes that facilitate cyst enlargement are hypothesized to result from two specific cellular abnormalities: (1) increased fluid secretion into the cyst lumen and (2) inappropriately increased cell division by the epithelium lining the cyst. Since there is no clinically approved specific or targeted therapy, current practice focuses on blood pressure control and statin therapy to reduce the cardiac mortality associated with chronic kidney disease. However, recent advances in our understanding of the pathways that govern renal cystogenesis have led to a number of intriguing possibilities in regard to therapeutic interventions. The purpose of this article is to review the pathogenesis of renal cyst formation and to review novel targets for the treatment of ADPKD.     

Bardet-Biedl syndrome highlights the major role of the primary cilium in efficient water reabsorption

Studies of the primary cilium, now known to be present in all cells, have undergone a revolution, in part, because mutation of many of its proteins causes a large number of diseases, including cystic kidney disease. Bardet-Biedl syndrome (BBS) is an inherited ciliopathy characterized, among other dysfunctions, by renal defects for which the precise role of the cilia in kidney function remains unclear. We studied a cohort of patients with BBS where we found that these patients had a urinary concentration defect even when kidney function was near normal and in the absence of major cyst formation. Subsequent in vitro analysis showed that renal cells in which a BBS gene was knocked down were unciliated, but did not exhibit cell cycle defects. As the vasopressin receptor 2 is located in the primary cilium, we studied BBS-derived unciliated renal epithelial cells and found that they were unable to respond to luminal arginine vasopressin treatment and activate their luminal aquaporin 2. The ability to reabsorb water was restored by treating these unciliated renal epithelial cells with forskolin, a receptor-independent adenylate cyclase activator, showing that the intracellular machinery for water absorption was present but not activated. These findings suggest that the luminal receptor located on the primary cilium may be important for efficient transepithelial water absorption.     

Cilium-generated signaling: a cellular GPS?

PURPOSE OF REVIEW: The discovery of the importance of proteins localized to cilia and basal bodies has provided novel insights into the pathogenesis of various human disorders including cystic kidney disease. The physiological role of cilium-generated signaling in most tissues including the kidney has remained elusive, however. This review focuses on the most recent advance in understanding a role for signaling through cilia for kidney biology. RECENT FINDINGS: Recent work has tied the function of several developmental master regulators to cilium-generated signaling in vertebrates. Hedgehog signaling requires ciliary proteins and loss of targeting of its receptors and effectors to cilia abrogates Hedgehog function. Moreover, the ciliary protein inversin has been shown to act as a molecular switch for the regulation of Wnt signaling cascades. SUMMARY: Together with recent data on a function of cilia and basal body proteins in planar cell polarity in mice, a novel concept and testable hypothesis for the function of cilia in vertebrates is emerging: cilia may act as a cellular positioning device to allow subcellular asymmetry and polarization of the cells within the plane of the epithelium to develop and maintain regular arrays of cells such as kidney tubes.     

The polycystic kidney disease proteins,polycystin-1, polycystin-2, polaris,and cystin,are co-localized in renal cilia

Recent evidence has suggested an association between structural and/or functional defects in the primary apical cilium of vertebrate epithelia and polycystic kidney disease (PKD). In Caenorhabditis elegans, the protein orthologues of the PKD-related proteins, polycystin-1 (LOV-1), polycystin-2 (PKD2), and polaris (OSM-5), co-localize in the cilia of male-specific sensory neurons, and defects in these proteins cause abnormalities of cilia structure and/or function. This study sought to determine whether the mammalian polycystins are expressed in primary cilia of renal epithelia and whether these proteins co-localize with polaris and cystin, the newly described, cilia-associated protein that is disrupted in the cpk mouse. To begin to address this issue, the expression of the protein products encoded by the PKD1, PKD2, Tg737, and cpk genes were examined in mouse cortical collecting duct (mCCD) cells using an immunofluorescence-based approach with a series of previously well-characterized antibodies. The mCCD cells were grown on cell culture inserts to optimize cell polarization and cilia formation. The data demonstrate co-localization in cilia of polycystin-1 and polycystin-2, which are the principal proteins involved in autosomal dominant polycystic kidney disease, with polaris and cystin, which are proteins that are disrupted in the Tg737(orpk)and cpk mouse models of autosomal recessive polycystic kidney disease, respectively. These data add to a growing body of evidence that suggests that primary cilium plays a key role in normal physiologic functions of renal epithelia and that defects in ciliary function contribute to the pathogenesis of PKD.