The Role of Extracellular Vesicles in SARS-CoV-2-Induced Acute Kidney Injury: An Overview.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions worldwide since its outbreak in the winter of 2019. While extensive research has primarily focused on the deleterious respiratory effects of SARS-CoV-2 in recent years, its pan-tropism has become evident. Among the vital organs susceptible to SARS-CoV-2 infection is the kidney. Post SARS-CoV-2 infection, patients have developed coronavirus disease 19 (COVID-19), with reported incidences of COVID-19 patients developing acute kidney injury (AKI). Given COVID-19's multisystemic manifestation, our review focuses on the impact of SARS-CoV-2 infection within the renal system with an emphasis on the current hypotheses regarding the role of extracellular vesicles (EVs) in SARS-CoV-2 pathogenesis. Emerging studies have shown that SARS-CoV-2 can directly infect the kidney, whereas EVs are involved in the spreading of SARS-CoV-2 particles to other neighboring cells. Once the viral particles are within the kidney system, many proinflammatory signaling pathways are shown to be activated, resulting in AKI. Hence, clinical investigation of urinary proinflammatory components and total urinary extracellular vesicles (uEVs) with viral particles have been used to assess the severity of AKI in patients with COVID-19. Remarkedly, new emerging studies have shown the potential of mesenchymal stem cell-derived EVs (MSC-EVs) and ACE2-containing EVs as a hopeful therapeutic tool to inhibit SARS-CoV-2 RNA replication and block viral entry, respectively. Overall, understanding EVs' physiological role is crucial and hopefully will rejuvenate our therapeutic approach towards COVID-19 patients with AKI.

Folate conjugated nanomedicines for selective inhibition of mTOR signaling in polycystic kidneys at clinically relevant doses.

Although rapamycin is a very effective drug for rodents with polycystic kidney disease (PKD), it is not encouraging in the clinical trials due to the suboptimal dosages compelled by the off-target side effects. We here report the generation, characterization, specificity, functionality, pharmacokinetic, pharmacodynamic and toxicology profiles of novel polycystic kidney-specific-targeting nanoparticles (NPs). We formulated folate-conjugated PLGA-PEG NPs, which can be loaded with multiple drugs, including rapamycin (an mTOR inhibitor) and antioxidant 4-hydroxy-TEMPO (a nephroprotective agent). The NPs increased the efficacy, potency and tolerability of rapamycin resulting in an increased survival rate and improved kidney function by decreasing side effects and reducing biodistribution to other organs in PKD mice. The daily administration of rapamycin-alone (1 mg/kg/day) could now be achieved with a weekly injection of NPs containing rapamycin (379 µg/kg/week). This polycystic kidney-targeting nanotechnology, for the first time, integrated advances in the use of 1) nanoparticles as a delivery cargo, 2) folate for targeting, 3) near-infrared Cy5-fluorophore for in vitro and in vivo live imaging, 4) rapamycin as a pharmacological therapy, and 5) TEMPO as a combinational therapy. The slow sustained-release of rapamycin by polycystic kidney-targeting NPs demonstrates a new era of nanomedicine in treatment for chronic kidney diseases at clinically relevant doses.

The use of advanced spectral imaging to reveal nanoparticle identity in biological samples.

Nanoparticles (NPs) have been used in drug delivery therapies, medical diagnostic strategies, and as current Covid-19 vaccine carriers. Many microscope-based imaging systems have been introduced to facilitate detection and visualization of NPs. Unfortunately, none can differentiate the core and the shell of NPs. Spectral imaging has been used to distinguish a drug molecule and its metabolite. We have recently integrated this technology to a resolution of 9 nm by using artificial intelligence-driven analyses. Such a resolution allowed us to collect many robust datapoints for each pixel of an image. Our analyses could recognize 45 spectral points within a pixel to detect unlabeled Ag-NPs and Au-NPs in single live cells and tissues (liver, heart, spleen and kidneys). The improved resolution and software provided a more specific fingerprinting for each single molecule, allowing simultaneous analyses of 990 complex interactions from the 45 points for each molecule within a pixel of an image. This in turn allowed us to detect surface-functionalization of Ag-NPs to distinguish the core from the shell of Ag-NPs for the first time. Our studies were validated using various laborious and time-consuming conventional techniques. We propose that spectral imaging has tremendous potential to study NP localization and identification in biological samples at a high temporal and spatial resolution, based primarily on spectral identity information.

Cilia proteins are biomarkers of altered flow in the vasculature.

Cilia, microtubule-based organelles that project from the apical luminal surface of endothelial cells (ECs), are widely regarded as low-flow sensors. Previous reports suggest that upon high shear stress, cilia on the EC surface are lost, and more recent evidence suggests that deciliation-the physical removal of cilia from the cell surface-is a predominant mechanism for cilia loss in mammalian cells. Thus, we hypothesized that EC deciliation facilitated by changes in shear stress would manifest in increased abundance of cilia-related proteins in circulation. To test this hypothesis, we performed shear stress experiments that mimicked flow conditions from low to high shear stress in human primary cells and a zebrafish model system. In the primary cells, we showed that upon shear stress induction, indeed, ciliary fragments were observed in the effluent in vitro, and effluents contained ciliary proteins normally expressed in both endothelial and epithelial cells. In zebrafish, upon shear stress induction, fewer cilia-expressing ECs were observed. To test the translational relevance of these findings, we investigated our hypothesis using patient blood samples from sickle cell disease and found that plasma levels of ciliary proteins were elevated compared with healthy controls. Further, sickled red blood cells demonstrated high levels of ciliary protein (ARL13b) on their surface after adhesion to brain ECs. Brain ECs postinteraction with sickle RBCs showed high reactive oxygen species (ROS) levels. Attenuating ROS levels in brain ECs decreased cilia protein levels on RBCs and rescued ciliary protein levels in brain ECs. Collectively, these data suggest that cilia and ciliary proteins in circulation are detectable under various altered-flow conditions, which could serve as a surrogate biomarker of the damaged endothelium.

Ciliary extracellular vesicles are distinct from the cytosolic extracellular vesicles.

Extracellular vesicles (EVs) are cell-derived membrane vesicles that are released into the extracellular space. EVs encapsulate key proteins and mediate intercellular signalling pathways. Recently, primary cilia have been shown to release EVs under fluid-shear flow, but many proteins encapsulated in these vesicles have never been identified. Primary cilia are ubiquitous mechanosensory organelles that protrude from the apical surface of almost all human cells. Primary cilia also serve as compartments for signalling pathways, and their defects have been associated with a wide range of human genetic diseases called ciliopathies. To better understand the mechanism of ciliopathies, it is imperative to know the distinctive protein profiles of the differently sourced EVs (cilia vs cytosol). Here, we isolated EVs from ciliated wild-type (WT) and non-ciliated IFT88 knockout (KO) mouse endothelial cells using fluid-shear flow followed by a conventional method of EV isolation. EVs isolated from WT and KO exhibited distinctive sizes. Differences in EV protein contents were studied using liquid chromatography with tandem mass spectrometry (LC-MS-MS) and proteomic comparative analysis, which allowed us to classify proteins between ciliary EVs and cytosolic EVs derived from WT and KO, respectively. A total of 79 proteins were exclusively expressed in WT EVs, 145 solely in KO EVs, and 524 in both EVs. Our bioinformatics analyses revealed 29% distinct protein classes and 75% distinct signalling pathways between WT and KO EVs. Based on our statistical analyses and in vitro studies, we identified NADPH-cytochrome P450 reductase (POR), and CD166 antigen (CD166) as potential biomarkers for ciliary and cytosolic EVs, respectively. Our protein-protein interaction network analysis revealed that POR, but not CD166, interacted with either established or strong ciliopathy gene candidates. This report shows the unique differences between EVs secreted from cilia and the cytosol. These results will be important in advancing our understanding of human genetic diseases.

Novel biomarkers of ciliary extracellular vesicles interact with ciliopathy and Alzheimer's associated proteins.

Ciliary extracellular vesicles (ciEVs), released from primary cilia, contain functional proteins that play an important role in cilia structure and functions. We have recently shown that ciEVs and cytosolic extracellular vesicles (cyEVs) have unique and distinct biomarkers. While ciEV biomarkers have shown some interactions with known ciliary proteins, little is known about the interaction of ciEV proteins with proteins involved in ciliopathy and neurodegenerative disorders. Here, we reveal for the first time the protein-protein interaction (PPI) between the top five ciEVs biomarkers with ciliopathy and Alzheimer disease (AD) proteins. These results support the growing evidence of the critical physiological roles of cilia in neurodegenerative disorders.

Proteomic Identification Reveals the Role of Ciliary Extracellular-Like Vesicle in Cardiovascular Function.

Primary cilia are shown to have membrane swelling, also known as ciliary bulbs. However, the role of these structures and their physiological relevance remains unknown. Here, it is reported that a ciliary bulb has extracellular vesicle (EV)-like characteristics. The ciliary extracellular-like vesicle (cELV) has a unique dynamic movement and can be released by mechanical fluid force. To better identify the cELV, differential multidimensional proteomic analyses are performed on the cELV. A database of 172 cELV proteins is generated, and all that examined are confirmed to be in the cELV. Repressing the expression of these proteins in vitro and in vivo inhibits cELV formation. In addition to the randomized heart looping, hydrocephalus, and cystic kidney in fish, compensated heart contractility is observed in both fish and mouse models. Specifically, low circulation of cELV results in hypotension with compensated heart function, left ventricular hypertrophy, cardiac fibrosis, and arrhythmogenic characteristics, which result in a high mortality rate in mice. Furthermore, the overall ejection fraction, stroke volume, and cardiac output are significantly decreased in mice lacking cELV. It is thus proposed that the cELV as a nanocompartment within a primary cilium plays an important role in cardiovascular functions.

Author Correction: Sensory primary cilium is a responsive cAMP microdomain in renal epithelia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Measurement of cytoplasmic and cilioplasmic calcium in a single living cell.

Cellular signaling represents an evolution of biological systems to sense external stimuli and communicate extracellular microenvironment to the intracellular compartments. The processes underlying molecular signaling have been widely studied due to their important cellular functions. There are numerous techniques available to quantitate the different molecules involved in cellular processes. Among them, calcium is a ubiquitous signaling molecule involved in many biological pathways. Over time the methods to measure intracellular calcium have advanced to better understand its role as a second messenger. In this chapter, we introduce a method to study a single cilium, a mechanosensor that elicits a calcium signaling cascade. To successfully observe the calcium changes in this thin cylindrical-like projection from the cell surface, we utilize a genetically encoded sensor with a high spatial and temporal resolution. In addition, the probe must be localized to the ciliary compartment in order to observe the intraciliary calcium signaling dynamics. To this end, a cilium targeting genetically encoded indicator is used to observe calcium fluxes in both cytoplasm and cilioplasm.

Sensory primary cilium is a responsive cAMP microdomain in renal epithelia.

Primary cilia are hair-like cellular extensions that sense microenvironmental signals surrounding cells. The role of adenylyl cyclases in ciliary function has been of interest because the product of adenylyl cyclase activity, cAMP, is relevant to cilia-related diseases. In the present study, we show that vasopressin receptor type-2 (V2R) is localized to cilia in kidney epithelial cells. Pharmacologic inhibition of V2R with tolvaptan increases ciliary length and mechanosensory function. Genetic knockdown of V2R, however, does not have any effect on ciliary length, although the effect of tolvaptan on ciliary length is dampened. Our study reveals that tolvaptan may have a cilia-specific effect independent of V2R or verapamil-sensitive calcium channels. Live-imaging of single cilia shows that V2R activation increases cilioplasmic and cytoplasmic cAMP levels, whereas tolvaptan mediates cAMP changes only in a cilia-specific manner. Furthermore, fluid-shear stress decreases cilioplasmic, but not cytoplasmic cAMP levels. Our data indicate that cilioplasmic and cytoplasmic cAMP levels are differentially modulated. We propose that the cilium is a critical sensor acting as a responsive cAMP microcompartment during physiologically relevant stimuli.

Ciliotherapy: Remote Control of Primary Cilia Movement and Function by Magnetic Nanoparticles.

Patients with polycystic kidney disease (PKD) are characterized with uncontrolled hypertension. Hypertension in PKD is a ciliopathy, an abnormal function and/or structure of primary cilia. Primary cilia are cellular organelles with chemo and mechanosensory roles. In the present studies, we designed a cilia-targeted (CT) delivery system to deliver fenoldopam specifically to the primary cilia. We devised the iron oxide nanoparticle (NP)-based technology for ciliotherapy. Live imaging confirmed that the CT-Fe2O3-NPs specifically targeted primary cilia in cultured cells in vitro and vascular endothelia in vivo. Importantly, the CT-Fe2O3-NPs enabled the remote control of the movement and function of a cilium with an external magnetic field, making the nonmotile cilium exhibit passive movement. The ciliopathic hearts displayed hypertrophy with compromised functions in left ventricle pressure, stroke volume, ejection fraction, and overall cardiac output because of prolonged hypertension. The CT-Fe2O3-NPs significantly improved cardiac function in the ciliopathic hypertensive models, in which the hearts also exhibited arrhythmia, which was corrected with the CT-Fe2O3-NPs. Intraciliary and cytosolic Ca2+ were increased when cilia were induced with fluid flow or magnetic field, and this served as a cilia-dependent mechanism of the CT-Fe2O3-NPs. Fenoldopam-alone caused an immediate decrease in blood pressure, followed by reflex tachycardia. Pharmacological delivery profiles confirmed that the CT-Fe2O3-NPs were a superior delivery system for targeting cilia more specifically, efficiently, and effectively than fenoldopam-alone. The CT-Fe2O3-NPs altered the mechanical properties of nonmotile cilia, and these nano-biomaterials had enormous clinical potential for ciliotherapy. Our studies further indicated that ciliotherapy provides a possibility toward personalized medicine in ciliopathy patients.

Personalized Nanotherapy by Specifically Targeting Cell Organelles To Improve Vascular Hypertension.

Ciliopathies caused by abnormal function of primary cilia include expanding spectrum of kidney, liver, and cardiovascular disorders. There is currently no treatment available for patients with cilia dysfunction. Therefore, we generated and compared two different (metal and polymer) cilia-targeted nanoparticle drug delivery systems (CTNDDS), CT-DAu-NPs and CT-PLGA-NPs, for the first time. These CTNDDS loaded with fenoldopam were further compared to fenoldopam-alone. Live-imaging of single-cell-single-cilium analysis confirmed that CTNDDS specifically targeted to primary cilia. While CTNDDS did not show any advantages over fenoldopam-alone in cultured cells in vitro, CTNDDS delivered fenoldopam more superior than fenoldopam-alone by eliminating the side effect of reflex tachycardia in murine models. Although slow infusion was required for fenoldopam-alone in mice, bolus injection was possible for CTNDDS. Though there were no significant therapeutic differences between CT-DAu-NPs and CT-PLGA-NPs, CT-PLGA-NPs tended to correct ciliopathy parameters closer to normal physiological levels, indicating CT-PLGA-NPs were better cargos than CT-DAu-NPs. Both CTNDDS showed no systemic adverse effect. In summary, our studies provided scientific evidence that existing pharmacological agent could be personalized with advanced nanomaterials to treat ciliopathy by targeting cilia without the need of generating new drugs.

Regulation of Polycystin-1 Function by Calmodulin Binding.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common genetic disease that leads to progressive renal cyst growth and loss of renal function, and is caused by mutations in the genes encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. The PC1/PC2 complex localizes to primary cilia and can act as a flow-dependent calcium channel in addition to numerous other signaling functions. The exact functions of the polycystins, their regulation and the purpose of the PC1/PC2 channel are still poorly understood. PC1 is an integral membrane protein with a large extracytoplasmic N-terminal domain and a short, ~200 amino acid C-terminal cytoplasmic tail. Most proteins that interact with PC1 have been found to bind via the cytoplasmic tail. Here we report that the PC1 tail has homology to the regulatory domain of myosin heavy chain including a conserved calmodulin-binding motif. This motif binds to CaM in a calcium-dependent manner. Disruption of the CaM-binding motif in PC1 does not affect PC2 binding, cilia targeting, or signaling via heterotrimeric G-proteins or STAT3. However, disruption of CaM binding inhibits the PC1/PC2 calcium channel activity and the flow-dependent calcium response in kidney epithelial cells. Furthermore, expression of CaM-binding mutant PC1 disrupts cellular energy metabolism. These results suggest that critical functions of PC1 are regulated by its ability to sense cytosolic calcium levels via binding to CaM.

Vascular Endothelial Primary Cilia: Mechanosensation and Hypertension.

Primary cilia are sensory organelles that extend from the cell surface and sense extracellular signals. Endothelial primary cilia protruding from the inner surface of blood vessel walls sense changes in blood flow and convert this mechanosensation into an intracellular biochemical/molecular signal, which triggers a cellular response. Primary endothelial cilia dysfunction may contribute to the impairment of this response and thus be directly implicated in the development of vascular abnormalities such as hypertension and aneurysms. Using both in vitro techniques as well as in vivo animal models, we and others have investigated fluid flow mechanosensory functions of endothelial cilia in cultured cells, animal models and autosomal dominant polycystic kidney disease (ADPKD) patients. More in-depth studies directed at identification of the mechanisms of fluid flow sensing will further enhance our knowledge of cilia-dependent vascular pathology. Although the current treatments aimed at treating the cardiovascular symptoms in ADPKD patients successfully slowed the progression of cyst growth, there is growing evidence which suggests that drugs which interfere with primary cilia function or structure could reduce cardiovascular complications in ADPKD. This review is to summarize the most recent studies on primary endothelial cilia function in the vascular system and to present primary cilia as a novel therapeutic target for vascular hypertension.

Chemical-Free Technique to Study the Ultrastructure of Primary Cilium.

A primary cilium is a hair-like structure with a width of approximately 200 nm. Over the past few decades, the main challenge in the study of the ultrastructure of cilia has been the high sensitivity of cilia to chemical fixation, which is required for many imaging techniques. In this report, we demonstrate a combined high-pressure freezing (HPF) and freeze-fracture transmission electron microscopy (FFTEM) technique to examine the ultrastructure of a cilium. Our objective is to develop an optimal high-resolution imaging approach that preserves cilia structures in their best natural form without alteration of cilia morphology by chemical fixation interference. Our results showed that a cilium has a swelling-like structure (termed bulb), which was previously considered a fixation artifact. The intramembrane particles observed via HPF/FFTEM indicated the presence of integral membrane proteins and soluble matrix proteins along the ciliary bulb, which is part of an integral structure within the ciliary membrane. We propose that HPF/FFTEM is an important and more suitable chemical-free method to study the ultrastructure of primary cilia.

Protein composition and movements of membrane swellings associated with primary cilia.

Dysfunction of many ciliary proteins has been linked to a list of diseases, from cystic kidney to obesity and from hypertension to mental retardation. We previously proposed that primary cilia are unique communication organelles that function as microsensory compartments that house mechanosensory molecules. Here we report that primary cilia exhibit membrane swellings or ciliary bulbs, which based on their unique ultrastructure and motility, could be mechanically regulated by fluid-shear stress. Together with the ultrastructure analysis of the swelling, which contains monosialodihexosylganglioside (GM3), our results show that ciliary bulb has a distinctive set of functional proteins, including GM3 synthase (GM3S), bicaudal-c1 (Bicc1), and polycystin-2 (PC2). In fact, results from our cilia isolation demonstrated for the first time that GM3S and Bicc1 are members of the primary cilia proteins. Although these proteins are not required for ciliary membrane swelling formation under static condition, fluid-shear stress induced swelling formation is partially modulated by GM3S. We therefore propose that the ciliary bulb exhibits a sensory function within the mechano-ciliary structure. Overall, our studies provided an important step towards understanding the ciliary bulb function and structure.

Ciliotherapy: a novel intervention in polycystic kidney disease.

BACKGROUND: Ciliopathies are a group of diseases associated with abnormal structure or function of primary cilia. Ciliopathies include polycystic kidney disease (PKD), a pathology associated with vascular hypertension. We previously showed that cilia length regulates cilia function, and cilia function is required for nitric oxide (NO) biosynthesis in endothelial cells. Because patients with PKD show abnormal sensory cilia function, the aim of our current study was to search for a targeted therapy focused on primary cilia, which we refer to as 'ciliotherapy'. METHODS AND RESULTS: In the present studies, our in vitro analyses refined fenoldopam as an equipotent and more specific dopaminergic agonist to regulate cilia length and function. Our in vivo studies indicated that fenoldopam increased cilia length and serum NO thereby reducing blood pressure in a PKD mouse model. Our crossover, multicenter, double-blind and placebo-controlled clinical study further indicated that cilia-targeting therapy showed an overall reduction in mean arterial pressure in PKD patients. CONCLUSIONS: Overall, our studies provide the first evidence of ciliotherapy as an innovative intervention in patients with abnormal primary cilia.

Cilioplasm is a cellular compartment for calcium signaling in response to mechanical and chemical stimuli.

Primary cilia with a diameter of ~200 nm have been implicated in development and disease. Calcium signaling within a primary cilium has never been directly visualized and has therefore remained a speculation. Fluid-shear stress and dopamine receptor type-5 (DR5) agonist are among the few stimuli that require cilia for intracellular calcium signal transduction. However, it is not known if these stimuli initiate calcium signaling within the cilium or if the calcium signal originates in the cytoplasm. Using an integrated single-cell imaging technique, we demonstrate for the first time that calcium signaling triggered by fluid-shear stress initiates in the primary cilium and can be distinguished from the subsequent cytosolic calcium response through the ryanodine receptor. Importantly, this flow-induced calcium signaling depends on the ciliary polycystin-2 calcium channel. While DR5-specific agonist induces calcium signaling mainly in the cilioplasm via ciliary CaV1.2, thrombin specifically induces cytosolic calcium signaling through the IP3 receptor. Furthermore, a non-specific calcium ionophore triggers both ciliary and cytosolic calcium responses. We suggest that cilia not only act as sensory organelles but also function as calcium signaling compartments. Cilium-dependent signaling can spread to the cytoplasm or be contained within the cilioplasm. Our study thus provides the first model to understand signaling within the cilioplasm of a living cell.

The Roles of Primary cilia in Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited genetic disorder that results in progressive renal cyst formation with ultimate loss of renal function and other systemic disorders. These systemic disorders include abnormalities in cardiovascular, portal, pancreatic and gastrointestinal systems. ADPKD is considered to be among the ciliopathy diseases due to the association with abnormal primary cilia function. In order to understand the full course of primary cilia and its association with ADPKD, the structure, functions and role of primary cilia have been meticulously investigated. As a result, the focus on primary cilia has emerged to support the vital roles of primary cilia in ADPKD. The primary cilia have been shown to have not only a mechanosensory function but also a chemosensory function. Both structural and functional defects in primary cilia result in cystic kidney disease and vascular hypertension. Thus, the mechanosenory and chemosensory functions will be analyzed in regards to ADPKD.