Real-Time Autophagic Flux Measurements in Live Cells Using a Novel Fluorescent Marker DAPRed.

Autophagy is a conserved homeostatic mechanism involved in cellular homeostasis and many disease processes. Although it was first described in yeast cells undergoing starvation, we have learned over the years that autophagy gets activated in many stress conditions and during development and aging in mammalian cells. Understanding the fundamental mechanisms underlying autophagy effects can bring us closer to better insights into the pathogenesis of many disease conditions (e.g., cardiac muscle necrosis, Alzheimer's disease, and chronic lung injury). Due to the complex and dynamic nature of the autophagic processes, many different techniques (e.g., western blotting, fluorescent labeling, and genetic modifications of key autophagy proteins) have been developed to delineate autophagy effects. Although these methods are valid, they are not well suited for the assessment of time-dependent autophagy kinetics. Here, we describe a novel approach: the use of DAPRed for autophagic flux measurement via live cell imaging, utilizing A549 cells, that can visualize and quantify autophagic flux in real time in single live cells. This approach is relatively straightforward in comparison to other experimental procedures and should be applicable to any in vitro cell/tissue models. Key features • Allows real-time qualitative imaging of autophagic flux at single-cell level. • Primary cells and cell lines can also be utilized with this technique. • Use of confocal microscopy allows visualization of autophagy without disturbing cellular functions.

Kinetics of autophagic activity in nanoparticle-exposed lung adenocarcinoma (A549) cells.

Autophagy, a homeostatic mechanism, is crucial in maintaining normal cellular function. Although dysregulation of autophagic processes is recognized in certain diseases, it is unknown how maintenance of cellular homeostasis might be affected by the kinetics of autophagic activity in response to various stimuli. In this study, we assessed those kinetics in lung adenocarcinoma (A549) cells in response to exposure to nanoparticles (NP) and/or Rapamycin. Since NP are known to induce autophagy, we wished to determine if this phenomenon could be a driver of the harmful effects seen in lung tissues exposed to air pollution. A549 cells were loaded with a fluorescent marker (DAPRed) that labels autophagosomes and autolysosomes. Autophagic activity was assessed based on the fluorescence intensity of DAPRed measured over the entire cell volume of live single cells using confocal laser scanning microscopy (CLSM). Autophagic activity over time was determined during exposure of A549 cells to single agents (50 nM Rapamycin; 80 µg/mL, 20 nm carboxylated polystyrene NP (PNP); or, 1 µg/mL ambient ultrafine particles (UFP) (<180 nm)), or double agents (Rapamycin + PNP or Rapamycin + UFP; concomitant and sequential), known to stimulate autophagy. Autophagic activity increased in all experimental modalities, including both single agent and double agent exposures, and reached a steady state in all cases ~2 times control from ~8 to 24 hrs, suggesting the presence of an upper limit to autophagic capacity. These results are consistent with the hypothesis that environmental stressors might exert their harmful effects, at least in part, by limiting available autophagic response to additional stimulation, thereby making nanoparticle-exposed cells more susceptible to secondary injury due to autophagic overload.

Biokinetic modeling of nanoparticle interactions with lung alveolar epithelial cells: uptake, intracellular processing, and egress.

Studies on health effects of engineered nanomaterials (ENMs) in the lung have provided information on ENM toxicity and translocation across airway and alveolar epithelial barriers. Various inhaled ENMs (e.g., gold and iridium nanoparticles) have been reported to partially cross the air-blood barrier in the lung, enter the vasculature, and distribute in several end organs, including the heart, liver, spleen, and kidney. Using an in vitro primary rat alveolar epithelial cell (AEC) monolayer model, we reported transport rates of relatively nontoxic polystyrene nanoparticles (PNPs), which appear to be taken up via nonendocytic processes into AECs. PNPs internalized into cytoplasm then trigger autophagy, followed by delivery of PNPs from autophagosomes into lysosomes, from where PNPs are exocytosed. We used the data from these experiments to perform biokinetic modeling that incorporates the processes associated with internalization and intracellular distribution of PNPs, autophagy, lysosomal exocytosis of PNPs, and several putative mechanisms of action that extend our previous understanding of AEC processing of PNPs. Results suggest that entry of PNPs into AECs, subsequent activation of autophagy by cytosolic PNPs, accumulation of PNPs in lysosomes, and lysosomal exocytosis are interwoven by proposed regulatory mechanisms.

Evidence for Nanoparticle-Induced Lysosomal Dysfunction in Lung Adenocarcinoma (A549) Cells.

BACKGROUND: Polystyrene nanoparticles (PNP) are taken up by primary rat alveolar epithelial cell monolayers (RAECM) in a time-, dose-, and size-dependent manner without involving endocytosis. Internalized PNP in RAECM activate autophagy, are delivered to lysosomes, and undergo [Ca2+]-dependent exocytosis. In this study, we explored nanoparticle (NP) interactions with A549 cells. METHODS: After exposure to PNP or ambient pollution particles (PM0.2), live single A549 cells were studied using confocal laser scanning microscopy. PNP uptake and egress were investigated and activation of autophagy was confirmed by immunolabeling with LC3-II and LC3-GFP transduction/colocalization with PNP. Mitochondrial membrane potential, mitophagy, and lysosomal membrane permeability (LMP) were assessed in the presence/absence of apical nanoparticle (NP) exposure. RESULTS: PNP uptake into A549 cells decreased in the presence of cytochalasin D, an inhibitor of macropinocytosis. PNP egress was not affected by increased cytosolic [Ca2+]. Autophagy activation was indicated by increased LC3 expression and LC3-GFP colocalization with PNP. Increased LMP was observed following PNP or PM0.2 exposure. Mitochondrial membrane potential was unchanged and mitophagy was not detected after NP exposure. CONCLUSIONS: Interactions between NP and A549 cells involve complex cellular processes leading to lysosomal dysfunction, which may provide opportunities for improved nanoparticle-based therapeutic approaches to lung cancer management.

Alveolar epithelial cell processing of nanoparticles activates autophagy and lysosomal exocytosis.

Using confocal microscopy, we quantitatively assessed uptake, processing, and egress of near-infrared (NIR)-labeled carboxylated polystyrene nanoparticles (PNP) in live alveolar epithelial cells (AEC) during interactions with primary rat AEC monolayers (RAECM). PNP fluorescence intensity (content) and colocalization with intracellular vesicles in a cell were determined over the entire cell volume via z stacking. Isotropic cuvette-based microfluorimetry was used to determine PNP concentration ([PNP]) from anisotropic measurements of PNP content assessed by confocal microscopy. Results showed that PNP uptake kinetics and steady-state intracellular content decreased as diameter increased from 20 to 200 nm. For 20-nm PNP, uptake rate and steady-state intracellular content increased with increased apical [PNP] but were unaffected by inhibition of endocytic pathways. Intracellular PNP increasingly colocalized with autophagosomes and/or lysosomes over time. PNP egress exhibited fast Ca2+ concentration-dependent release and a slower diffusion-like process. Inhibition of microtubule polymerization curtailed rapid PNP egress, resulting in elevated vesicular and intracellular PNP content. Interference with autophagosome formation led to slower PNP uptake and markedly decreased steady-state intracellular content. At steady state, cytosolic [PNP] was higher than apical [PNP], and vesicular [PNP] (~80% of intracellular PNP content) exceeded both cytosolic and intracellular [PNP]. These data are consistent with the following hypotheses: 1) autophagic processing of nanoparticles is essential for maintenance of AEC integrity; 2) altered autophagy and/or lysosomal exocytosis may lead to AEC injury; and 3) intracellular [PNP] in AEC can be regulated, suggesting strategies for enhancement of nanoparticle-driven AEC gene/drug delivery and/or amelioration of AEC nanoparticle-related cellular toxicity.

The macula densa prorenin receptor is essential in renin release and blood pressure control.

The prorenin receptor (PRR) was originally proposed to be a member of the renin-angiotensin system (RAS); however, recent work questioned their association. The present paper describes a functional link between the PRR and RAS in the renal juxtaglomerular apparatus (JGA), a classic anatomical site of the RAS. PRR expression was found in the sensory cells of the JGA, the macula densa (MD), and immunohistochemistry-localized PRR to the MD basolateral cell membrane in mouse, rat, and human kidneys. MD cell PRR activation led to MAP kinase ERK1/2 signaling and stimulation of PGE2 release, the classic pathway of MD-mediated renin release. Exogenous renin or prorenin added to the in vitro microperfused JGA-induced acute renin release, which was inhibited by removing the MD or by the administration of a PRR decoy peptide. To test the function of MD PRR in vivo, we established a new mouse model with inducible conditional knockout (cKO) of the PRR in MD cells based on neural nitric oxide synthase-driven Cre-lox recombination. Deletion of the MD PRR significantly reduced blood pressure and plasma renin. Challenging the RAS by low-salt diet + captopril treatment caused further significant reductions in blood pressure, renal renin, cyclooxygenase-2, and microsomal PGE synthase expression in cKO vs. wild-type mice. These results suggest that the MD PRR is essential in a novel JGA short-loop feedback mechanism, which is integrated within the classic MD mechanism to control renin synthesis and release and to maintain blood pressure.

Knockout mice reveal key roles for claudin 18 in alveolar barrier properties and fluid homeostasis.

Claudin proteins are major constituents of epithelial and endothelial tight junctions (TJs) that regulate paracellular permeability to ions and solutes. Claudin 18, a member of the large claudin family, is highly expressed in lung alveolar epithelium. To elucidate the role of claudin 18 in alveolar epithelial barrier function, we generated claudin 18 knockout (C18 KO) mice. C18 KO mice exhibited increased solute permeability and alveolar fluid clearance (AFC) compared with wild-type control mice. Increased AFC in C18 KO mice was associated with increased ß-adrenergic receptor signaling together with activation of cystic fibrosis transmembrane conductance regulator, higher epithelial sodium channel, and Na-K-ATPase (Na pump) activity and increased Na-K-ATPase ß1 subunit expression. Consistent with in vivo findings, C18 KO alveolar epithelial cell (AEC) monolayers exhibited lower transepithelial electrical resistance and increased solute and ion permeability with unchanged ion selectivity. Claudin 3 and claudin 4 expression was markedly increased in C18 KO mice, whereas claudin 5 expression was unchanged and occludin significantly decreased. Microarray analysis revealed changes in cytoskeleton-associated gene expression in C18 KO mice, consistent with observed F-actin cytoskeletal rearrangement in AEC monolayers. These findings demonstrate a crucial nonredundant role for claudin 18 in the regulation of alveolar epithelial TJ composition and permeability properties. Increased AFC in C18 KO mice identifies a role for claudin 18 in alveolar fluid homeostasis beyond its direct contributions to barrier properties that may, at least in part, compensate for increased permeability.

Nanoparticle translocation across mouse alveolar epithelial cell monolayers: species-specific mechanisms.

Studies of polystyrene nanoparticle (PNP) trafficking across mouse alveolar epithelial cell monolayers (MAECM) show apical-to-basolateral flux of 20 and 120nm amidine-modified PNP is ~65 times faster than that of 20 and 100nm carboxylate-modified PNP, respectively. Calcium chelation with EGTA has little effect on amidine-modified PNP flux, but increases carboxylate-modified PNP flux ~50-fold. PNP flux is unaffected by methyl-ß-cyclodextrin, while ~70% decrease in amidine- (but not carboxylate-) modified PNP flux occurs across chlorpromazine- or dynasore-treated MAECM. Confocal microscopy reveals intracellular amidine- and carboxylate-modified PNP and association of amidine- (but not carboxylate-) modified PNP with clathrin heavy chain. These data indicate (1) amidine-modified PNP translocate across MAECM primarily via clathrin-mediated endocytosis and (2) physicochemical properties (e.g., surface charge) determine PNP interactions with mouse alveolar epithelium. Uptake/trafficking of nanoparticles into/across epithelial barriers is dependent on both nanoparticle physicochemical properties and (based on comparison with our prior results) specific epithelial cell type. FROM THE CLINICAL EDITOR: In this study of polystyrene nanoparticle trafficking across mouse alveolar epithelial cell monolayers, the authors determined that uptake/trafficking of nanoparticles into/across epithelial barriers is dependent on both nanoparticle physicochemical properties and the specific type of epithelial cells.

Olfactory receptor responding to gut microbiota-derived signals plays a role in renin secretion and blood pressure regulation.

Olfactory receptors are G protein-coupled receptors that mediate olfactory chemosensation and serve as chemosensors in other tissues. We find that Olfr78, an olfactory receptor expressed in the kidney, responds to short chain fatty acids (SCFAs). Olfr78 is expressed in the renal juxtaglomerular apparatus, where it mediates renin secretion in response to SCFAs. In addition, both Olfr78 and G protein-coupled receptor 41 (Gpr41), another SCFA receptor, are expressed in smooth muscle cells of small resistance vessels. Propionate, a SCFA shown to induce vasodilation ex vivo, produces an acute hypotensive response in wild-type mice. This effect is differentially modulated by disruption of Olfr78 and Gpr41 expression. SCFAs are end products of fermentation by the gut microbiota and are absorbed into the circulation. Antibiotic treatment reduces the biomass of the gut microbiota and elevates blood pressure in Olfr78 knockout mice. We conclude that SCFAs produced by the gut microbiota modulate blood pressure via Olfr78 and Gpr41.

A novel dual-color reporter for identifying insulin-producing beta-cells and classifying heterogeneity of insulinoma cell lines.

Many research studies use immortalized cell lines as surrogates for primary beta- cells. We describe the production and use of a novel "indirect" dual-fluorescent reporter system that leads to mutually exclusive expression of EGFP in insulin-producing (INS(+)) beta-cells or mCherry in non-beta-cells. Our system uses the human insulin promoter to initiate a Cre-mediated shift in reporter color within a single transgene construct and is useful for FACS selection of cells from single cultures for further analysis. Application of our reporter to presumably clonal HIT-T15 insulinoma cells, as well as other presumably clonal lines, indicates that these cultures are in fact heterogeneous with respect to INS(+) phenotype. Our strategy could be easily applied to other cell- or tissue-specific promoters. We anticipate its utility for FACS purification of INS(+) and glucose-responsive beta-like-cells from primary human islet cell isolates or in vitro differentiated pluripotent stem cells.

Translocation of PEGylated quantum dots across rat alveolar epithelial cell monolayers.

BACKGROUND: In this study, primary rat alveolar epithelial cell monolayers (RAECM) were used to investigate transalveolar epithelial quantum dot trafficking rates and underlying transport mechanisms. METHODS: Trafficking rates of quantum dots (PEGylated CdSe/ZnS, core size 5.3 nm, hydrodynamic size 25 nm) in the apical-to-basolateral direction across RAECM were determined. Changes in bioelectric properties (ie, transmonolayer resistance and equivalent active ion transport rate) of RAECM in the presence or absence of quantum dots were measured. Involvement of endocytic pathways in quantum dot trafficking across RAECM was assessed using specific inhibitors (eg, methyl-ß-cyclodextrin, chlorpromazine, and dynasore for caveolin-, clathrin-, and dynamin-mediated endocytosis, respectively). The effects of lowering tight junctional resistance on quantum dot trafficking were determined by depleting Ca(2+) in apical and basolateral bathing fluids of RAECM using 2 mM EGTA. Effects of temperature on quantum dot trafficking were studied by lowering temperature from 37°C to 4°C. RESULTS: Apical exposure of RAECM to quantum dots did not elicit changes in transmonolayer resistance or ion transport rate for up to 24 hours; quantum dot trafficking rates were not surface charge-dependent; methyl-ß-cyclodextrin, chlorpromazine, and dynasore did not decrease quantum dot trafficking rates; lowering of temperature decreased transmonolayer resistance by approximately 90% with a concomitant increase in quantum dot trafficking by about 80%; and 24 hours of treatment of RAECM with EGTA decreased transmonolayer resistance by about 95%, with increased quantum dot trafficking of up to approximately 130%. CONCLUSION: These data indicate that quantum dots do not injure RAECM and that quantum dot trafficking does not appear to take place via endocytic pathways involving caveolin, clathrin, or dynamin. We conclude that quantum dot translocation across RAECM takes place via both transcellular and paracellular pathways and, based on comparison with our prior studies, interactions of nanoparticles with RAECM are strongly dependent on nanoparticle composition and surface properties.

Nanomaterial interactions with and trafficking across the lung alveolar epithelial barrier: implications for health effects of air-pollution particles.

Studies on the health effects of air-pollution particles suggest that injury may result from inhalation of airborne ultrafine particles (<100 nm in diameter). Engineered nanomaterials (<100 nm in at least one dimension) may also be harmful if inhaled. Nanomaterials deposited on the respiratory epithelial tract are thought to cross the air-blood barrier, especially via the expansive alveolar region, into the systemic circulation to reach end organs (e.g., myocardium, liver, pancreas, kidney, and spleen). Since ambient ultrafine particles are difficult to track, studies of defined engineered nanomaterials have been used to obtain valuable information on how nanomaterials interact with and traffic across the air-blood barrier of mammalian lungs. Since specific mechanistic information on how nanomaterials interact with the lung is difficult to obtain using in vivo or ex vivo lungs due to their complex anatomy, in vitro alveolar epithelial models have been of considerable value in determining nanomaterial-lung interactions. In this review, we provide information on mechanisms underlying lung alveolar epithelial injury caused by various nanomaterials and on nanomaterial trafficking across alveolar epithelium that may lead to end-organ injury.

Direct demonstration of tubular fluid flow sensing by macula densa cells.

Macula densa (MD) cells in the cortical thick ascending limb (cTAL) detect variations in tubular fluid composition and transmit signals to the afferent arteriole (AA) that control glomerular filtration rate [tubuloglomerular feedback (TGF)]. Increases in tubular salt at the MD that normally parallel elevations in tubular fluid flow rate are well accepted as the trigger of TGF. The present study aimed to test whether MD cells can detect variations in tubular fluid flow rate per se. Calcium imaging of the in vitro microperfused isolated JGA-glomerulus complex dissected from mice was performed using fluo-4 and fluorescence microscopy. Increasing cTAL flow from 2 to 20 nl/min (80 mM [NaCl]) rapidly produced significant elevations in cytosolic Ca(2+) concentration ([Ca(2+)](i)) in AA smooth muscle cells [evidenced by changes in fluo-4 intensity (F); F/F(0) = 1.45 ± 0.11] and AA vasoconstriction. Complete removal of the cTAL around the MD plaque and application of laminar flow through a perfusion pipette directly to the MD apical surface essentially produced the same results even when low (10 mM) or zero NaCl solutions were used. Acetylated α-tubulin immunohistochemistry identified the presence of primary cilia in mouse MD cells. Under no flow conditions, bending MD cilia directly with a micropipette rapidly caused significant [Ca(2+)](i) elevations in AA smooth muscle cells (fluo-4 F/F(0): 1.60 ± 0.12) and vasoconstriction. P2 receptor blockade with suramin significantly reduced the flow-induced TGF, whereas scavenging superoxide with tempol did not. In conclusion, MD cells are equipped with a tubular flow-sensing mechanism that may contribute to MD cell function and TGF.

A high-powered view of the filtration barrier.

Multiphoton excitation fluorescence microscopy is a powerful noninvasive imaging technique for the deep optical sectioning of living tissues. Its application in several intact tissues is a significant advance in our understanding of organ function, including renal pathophysiological mechanisms. The glomerulus, the filtering unit in the kidney, is one good example of a relatively inaccessible and complex structure, with cell types that are otherwise difficult to study at high resolution in their native environment. In this article, we address the application, advantages, and limitations of this imaging technology for the study of the glomerular filtration barrier and the controversy it recently generated regarding the glomerular filtration of macromolecules. More advanced and accurate multiphoton determinations of the glomerular sieving coefficient that are presented here dismiss previous claims on the filtration of nephrotic levels of albumin. The sieving coefficient of 70-kD dextran was found to be around 0.001. Using a model of focal segmental glomerulosclerosis, increased filtration barrier permeability is restricted only to areas of podocyte damage, consistent with the generally accepted role of podocytes and the glomerular origin of albuminuria. Time-lapse imaging provides new details and important in vivo confirmation of the dynamics of podocyte movement, shedding, replacement, and the role of the parietal epithelial cells and Bowman's capsule in the pathology of glomerulosclerosis.

Loss of renal microvascular integrity in postnatal Crim1 hypomorphic transgenic mice.

Crim1 is a cell-surface, transmembrane protein that binds to a variety of cystine knot-containing growth factors, including vascular endothelial growth factor A. In the developing renal glomerulus, Crim1 acts to tether vascular endothelial growth factor A to the podocyte cell surface, thus regulating its release to glomerular endothelial cells. The hypomorphic transgenic mouse (Crim1(KST264/KST264)) has glomerular cysts and severe glomerular vascular defects because of the lack of functional Crim1 in the glomerulus. Adult transgenic mice have a reduced glomerular filtration rate and glomerular capillary defects. We now show that, in these adult transgenic mice, renal vascular defects are not confined to the glomerulus but also extend to the peritubular microvasculature, as live imaging revealed leakiness of both glomerular and peritubular capillaries. An ultrastructural analysis of the microvasculature showed an abnormal endothelium and collagen deposition between the endothelium and the tubular basement membrane, present even in juvenile mice. Overt renal disease, including fibrosis and renin recruitment, was not evident until adulthood. Our study suggests that Crim1 is involved in endothelial maintenance and integrity and its loss contributes to a primary defect in the extraglomerular vasculature.

Connexin 30 deficiency impairs renal tubular ATP release and pressure natriuresis.

In the renal tubule, ATP is an important regulator of salt and water reabsorption, but the mechanism of ATP release is unknown. Several connexin (Cx) isoforms form mechanosensitive, ATP-permeable hemichannels. We localized Cx30 to the nonjunctional apical membrane of cells in the distal nephron and tested whether Cx30 participates in physiologically important release of ATP. We dissected, partially split open, and microperfused cortical collecting ducts from wild-type and Cx30-deficient mice in vitro. We used PC12 cells as ATP biosensors by loading them with Fluo-4/Fura Red to measure cytosolic calcium and positioning them in direct contact with the apical surface of either intercalated or principal cells. ATP biosensor responses, triggered by increased tubular flow or by bath hypotonicity, were approximately three-fold greater when positioned next to intercalated cells than next to principal cells. In addition, these responses did not occur in preparations from Cx30-deficient mice or with purinergic receptor blockade. After inducing step increases in mean arterial pressure by ligating the distal aorta followed by the mesenteric and celiac arteries, urine output increased 4.2-fold in wild-type mice compared with 2.6-fold in Cx30-deficient mice, and urinary Na(+) excretion increased 5.2-fold in wild-type mice compared with 2.8-fold in Cx30-deficient mice. Furthermore, Cx30-deficient mice developed endothelial sodium channel-dependent, salt-sensitive elevations in mean arterial pressure. Taken together, we suggest that mechanosensitive Cx30 hemichannels have an integral role in pressure natriuresis by releasing ATP into the tubular fluid, which inhibits salt and water reabsorption.

Multiphoton imaging of renal regulatory mechanisms.

Most physiological functions of the kidneys, including the clearance of metabolic waste products, maintenance of body fluid, electrolyte homeostasis, and blood pressure, are achieved by complex interactions between multiple renal cell types and previously inaccessible structures in many organ parts that have been difficult to study. Multiphoton fluorescence microscopy offers a state-of-the-art imaging technique for deep optical sectioning of living tissues and organs with minimal deleterious effects. Dynamic regulatory processes and multiple functions in the intact kidney can be quantitatively visualized in real time, noninvasively, and with submicron resolution. This article reviews innovative multiphoton imaging technologies and their applications that provided the most complex, immediate, and dynamic portrayal of renal function-clearly depicting as well as analyzing the components and mechanisms involved in renal (patho)physiology.

From in vitro to in vivo: imaging from the single cell to the whole organism.

This unit addresses the applications of fluorescence microscopy and quantitative imaging to study multiple physiological variables of living tissue. Protocols are presented for fluorescence-based investigations ranging from in vitro cell and tissue approaches to in vivo imaging of intact organs. These include the measurement of cytosolic parameters both in vitro and in vivo (such as calcium, pH, and nitric oxide), dynamic cellular processes (renin granule exocytosis), FRET-based real-time assays of enzymatic activity (renin), physiological processes (vascular contraction, membrane depolarization), and whole organ functional parameters (blood flow, glomerular filtration). Multi-photon microscopy is ideal for minimally invasive and undisruptive deep optical sectioning of the living tissue, which translates into ultra-sensitive real-time measurement of these parameters with high spatial and temporal resolution. With the combination of cell and tissue cultures, microperfusion techniques, and whole organ or animal models, fluorescence imaging provides unmatched versatility for biological and medical studies of the living organism.

Connexin45 is expressed in the juxtaglomerular apparatus and is involved in the regulation of renin secretion and blood pressure.

Connexin (Cx) proteins are known to play a role in cell-to-cell communication via intercellular gap junction channels or transiently open hemichannels. Previous studies have identified several connexin isoforms in the juxtaglomerular apparatus (JGA), but the vascular connexin isoform Cx45 has not yet been studied in this region. The present work aimed to identify in detail the localization of Cx45 in the JGA and to suggest a functional role for Cx45 in the kidney using conditions where Cx45 expression or function was altered. Using mice that express lacZ coding DNA under the control of the Cx45 promoter, we observed beta-galactosidase staining in cortical vasculature and glomeruli, with specific localization to the JGA region. Renal vascular localization of Cx45 was further confirmed with the use of conditional Cx45-deficient (Cx45fl/fl:Nestin-Cre) mice, which express enhanced green fluorescence protein (EGFP) instead of Cx45 only in cells that, during development, expressed the intermediate filament nestin. EGFP fluorescence was found in the afferent and efferent arteriole smooth muscle cells, in the renin-producing juxtaglomerular cells, and in the extra- and intraglomerular mesangium. Cx45fl/fl:Nestin-Cre mice exhibited increased renin expression and activity, as well as higher systemic blood pressure. The propagation of mechanically induced calcium waves was slower in cultured vascular smooth muscle cells (VSMCs) from Cx45fl/fl:Nestin-Cre mice and in control VSMC treated with a Cx45 gap mimetic peptide that inhibits Cx45 gap junctional communication. VSMCs allowed the cell-to-cell passage of the gap junction permeable dye Lucifer yellow, and calcium wave propagation was not altered by addition of the ATP receptor blocker suramin, suggesting that Cx45 regulates calcium wave propagation via direct gap junction coupling. In conclusion, the localization of Cx45 to the JGA and functional data from Cx45fl/fl:Nestin-Cre mice suggest that Cx45 is involved in the propagation of JGA vascular signals and in the regulation of renin release and blood pressure.

Succinate receptor GPR91 provides a direct link between high glucose levels and renin release in murine and rabbit kidney.

Diabetes mellitus is the most common and rapidly growing cause of end-stage renal disease in developed countries. A classic hallmark of early diabetes mellitus includes activation of the renin-angiotensin system (RAS), which may lead to hypertension and renal tissue injury, but the mechanism of RAS activation is elusive. Here we identified a paracrine signaling pathway in the kidney in which high levels of glucose directly triggered the release of the prohypertensive hormone renin. The signaling cascade involved the local accumulation of succinate and activation of the kidney-specific G protein-coupled metabolic receptor, GPR91, in the glomerular endothelium as observed in rat, mouse, and rabbit kidney sections. Elements of signal transduction included endothelial Ca2+, the production of NO and prostaglandin (PGE2), and their paracrine actions on adjacent renin-producing cells. This GPR91 signaling cascade may serve to modulate kidney function and help remove metabolic waste products through renal hyperfiltration, and it could also link metabolic diseases, such as diabetes, or metabolic syndrome with RAS overactivation, systemic hypertension, and organ injury.