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Rab10, a mammalian homolog of the yeast Sec4p protein, has previously been associated with endocytic recycling and biosynthetic membrane transport in cultured epithelia and with Glut4 translocation in adipocytes. Here, we report that Rab10 associates with primary cilia in renal epithelia in culture and in vivo. In addition, we find that Rab10 also colocalizes with exocyst proteins at the base of nascent cilia, and physically interacts with the exocyst complex, as detected with anti-Sec8 antibodies. These data suggest that membrane transport to the primary cilum may be mediated by interactions between Rab10 and an exocyst complex located at the cilium base. primary cilium; Sec8 doi: 10.1152/ajprenal.00198.2010.

Sulfonefluorescein (SF) is a fluorescent organic anion secreted by kidney proximal tubules. The purposes of this study were 1) to quantify accumulation of SF in normal and cystic rat kidneys in vivo and 2) to test whether SF accumulation could be used as a marker for cysts derived from proximal tubules. Male Munich-Wistar rats, normal Han:SPRD rats, and heterozygous Han:SPRD rats with autosomal-dominant polycystic kidney disease were anesthetized with Inactin and solutions containing SF were administered by constant intravenous infusion. In Munich-Wistar rats, SF fluorescence in the urinary space of Bowman's capsule averaged 0.15 [+ or -] 0.04 (n = 17) times that of glomerular capillary plasma, consistent with extensive plasma protein binding of SF. In normal Han:SPRD rats, steady-state cell cytoplasm SF fluorescence in proximal tubule and distal tubule cells averaged, respectively, 2.7 [+ or -] 1.4 (n = 99 tubules) and 0.2 [+ or -] 0.2 (n = 17) times that of peritubular capillary plasma. No punctate SF fluorescence was seen in proximal tubule cell cytoplasm. Probenecid reduced proximal tubule cell SF fluorescence to 0.64 [+ or -] 0.40 (n = 64) times that of plasma. Ureteral obstruction decreased the proximal tubule cell-to-lumen SF fluorescence gradient, suggesting that tubule fluid flow normally sweeps away secreted SF. In cystic kidneys, cysts derived from proximal tubules could be identified by their uptake of SF, but cell uptake was patchy. We conclude that in vivo two-photon microscopy is a powerful tool for quantifying glomerular and tubular handling of SF, and SF can be used to identify proximal tubule-derived cysts. organic anion secretion; glomerulus; polycystic kidney disease; two-photon microscopy

The ability to image calcium signals at subcellular levels within the intact depolarizing heart could provide valuable information toward a more integrated understanding of cardiac function. Accordingly, a system combining two-photon excitation with laser-scanning microscopy was developed to monitor electrically evoked [[Ca.sup.2+]].sub.i] transients in individual cardiomyocytes within noncontracting Langendorff-perfused mouse hearts. [[Ca.sup.2+]].sub.i] transients were recorded at depths [less than or equal to] 100 [micro]m from the epicardial surface with the fluorescent indicators rhod-2 or fura-2 in the presence of the excitation-contraction uncoupler cytochalasin D. Evoked [[Ca.sup.2+]].sub.i] transients were highly synchronized among neighboring cardiomyocytes. At 1 Hz, the times from 90 to 50% ([t.sub.90-50%]) and from 50 to 10% ([t.sub.50-10%]) of the peak [[Ca.sup.2+]].sub.i] were (means [+ or -] SE) 73 [+ or -] 4 and 126 [+ or -] 10 ms, respectively, and at 2 Hz, 62 [+ or -] 3 and 94 [+ or -] 6 ms (n = 19, P < 0.05 vs. 1 Hz) in rhod-2-loaded cardiomyocytes. [[Ca.sup.2+]].sub.i] decay was markedly slower in fura-2-loaded hearts ([t.sub.90-50%] at 1 Hz, 128 [+ or -] 9 ms and at 2 Hz, 88 [+ or -] 5 ms; [t.sub.50-10%] at 1 Hz, 214 [+ or -] 18 ms and at 2 Hz, 163 [+ or -] 7 ms; n = 19, P < 0.05 vs. rhod-2). Fura-2-induced deceleration of [[Ca.sup.2+]].sub.i] decline resulted from increased cytosolic [Ca.sup.2+] buffering, because the kinetics of rhod-2 decay resembled those obtained with fura-2 after incorporation of the [Ca.sup.2+] chelator BAPTA. Propagating calcium waves and [[Ca.sup.2+]].sub.i] amplitude alternans were readily detected in paced hearts. This approach should be of genera] utility to monitor the consequences of genetic and/or functional heterogeneity in cellular calcium signaling within whole mouse hearts at tissue depths that have been inaccessible to single-photon imaging. rhod-2; fura-2; BAPTA; 2,3-butanedione monoxime; cytochalasin D

Apoptosis is an important mode of cell death under both physiological and pathophysiological conditions. Numerous techniques are available for the study and quantitation of apoptosis in cell culture, but only few are useful when applied to complex tissues. Among these, the terminal transferase-mediated dUTP nick end-labeling (TUNEL) assay remains the most widely used technique. However, its specificity and sensitivity for the detection of apoptosis remain controversial. We developed a technique consisting of staining live cells and tissues with Hoechst 33342 and the vital dye propidium iodide (PI), followed by fixation and the TUNEL reaction. We demonstrate excellent retention of PI in necrotic cells after fixation. We also examined the distribution of TUNEL staining among necrotic and apoptotic cells in various models of cell injury in vitro and in vivo. We show that the sensitivity of the TUNEL varied between 61 and 90% in the models examined. The specificity exceeded 87% in all models but fell to 70% when a predominantly necrotic injury was induced. This novel and simple method will permit the determination of indices of sensitivity and specificity for the TUNEL assay in other tissues and experimental conditions. necrosis; propidium iodide; two-photon microscopy; ischemia

Optical microscopy, when applied to living animals, provides a powerful means of studying cell biology in the most physiologically relevant setting. The ability of two-photon microscopy to collect optical sections deep into biological tissues has opened up the field of intravital microscopy to high-resolution studies of the brain, lens, skin, and tumors. Here we present examples of the way in which two-photon microscopy can be applied to intravital studies of kidney physiology. Because the kidney is easily externalized without compromising its function, microscopy can be used to evaluate various aspects of renal function in vivo. These include cell vitality and apoptosis, fluid transport, receptor-mediated endocytosis, blood flow, and leukocyte trafficking. Efficient two-photon excitation of multiple fluorophores permits comparison of multiple probes and simultaneous characterization of multiple parameters and yields spectral information that is crucial to the interpretation of images containing uncharacterized autofluorescence. The studies described here demonstrate the way in which two-photon microscopy can provide a level of resolution previously unattainable in intravital microscopy, enabling kinetic analyses and physiological studies of the organs of living animals with subcellular resolution. fluorescence; multiphoton; in vivo

Rac1, but not RhoA, signaling protects epithelial adherens junction assembly during ATP depletion. Am J Physiol Cell Physiol 283: C261-C272, 2002; 10.1152/ ajpcell.00604.2001.--Rho family GTPase signaling regulates actin cytoskeleton and junctional complex assembly. Our previous work showed that RhoA signaling protects tight junctions from damage during ATP depletion. Here, we examined whether RhoA GTPase signaling protects adherens junction assembly during ATP depletion. Despite specific RhoA signaling- and ATP depletion-induced effects on adherens junction assembly, RhoA signaling did not alter adherens junction disassembly rates during ATP depletion. This shows that RhoA signaling specifically protects tight junctions from damage during ATP depletion. Rac1 GTPase signaling also regulates adherens junction assembly and therefore may regulate adherens junction assembly during ATP depletion. Indeed, we found that Rac1 signaling protects adherens junctions from damage during ATP depletion. Adherens junctions are regulated by various GTPases, including RhoA and Rac1, but adherens junctions are specifically protected by Rac1 signaling. ischemia; cadherin; catenin

Voxx: a PC-based, near real-time volume rendering system for biological microscopy. Am J Physiol Cell Physiol 282: C213-C218, 2002.--Confocal and two-photon fluorescence microscopy have advanced the exploration of complex, three-dimensional biological structures at submicron resolution. We have developed a voxel-based three-dimensional (3-D) imaging program (Voxx) capable of near real-time rendering that runs on inexpensive personal computers. This low-cost interactive 3-D imaging system provides a powerful tool for analyzing complex structures in cells and tissues and encourages a more thorough exploration of complex biological image data. confocal; three-dimensional; two-photon; voxels

The Indiana O'Brien Center for Advanced Microscopic Analysis is a National Institutes of Health (NIH) P30-funded research center dedicated to the development and dissemination of advanced methods of optical microscopy to support renal researchers throughout the world. The Indiana O'Brien Center was founded in 2002 as an NIH P-50 project with the original goal of helping researchers realize the potential of intravital multiphoton microscopy as a tool for understanding renal physiology and pathophysiology. The center has since expanded into the development and implementation of large-scale, high-content tissue cytometry. The advanced imaging capabilities of the center are made available to renal researchers worldwide via collaborations and a unique fellowship program. Center outreach is accomplished through an enrichment core that oversees a seminar series, an informational website, and a biennial workshop featuring hands-on training from members of the Indiana O'Brien Center and imaging experts from around the world. [ABSTRACT FROM AUTHOR]

Sandoval, Ruben M., Kenneth W. Dunn, and Bruce A. Molitoris. Gentamicin traffics rapidly and directly to the Golgi complex in LLC-[PK.sub.1] cells. Am J Physiol Renal Physiol 279: F884-F890, 2000.--To study the intracellular mechanisms of aminoglycoside toxicity, we used a 1:1 fluorescent conjugate of Texas Red and gentamicin (TRG) to quantify early uptake dynamics in renal epithelial (LLC-[PK.sub.1]) cells. Utilizing a protocol that quenches TRG fluorescence from lysosomes, the bulk of intracellular accumulation, we determined a portion rapidly trafficked directly to the Golgi complex when identified by a FITC-conjugated lectin from Lens culinaris agglutinin (LCA). A kinetic study over 120 min on cells showing total and quenched TRG fluorescence was then carried out, and the fluorescence intensity from the images was quantified. Trafficking of TRG to the Golgi complex occurred within 15 min and accounted for -20% of total cellular accumulation in the kinetic study. Colocalization studies using compartment-specific markers, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl sphingosine (C6NBD ceramide) and LCA, for the TGN trans-Golgi network, and the cis/medial-Golgi compartments, respectively, determined colocalization occurred with both Golgi compartments. These data support the existence of a pathway that directly and rapidly shuttles a portion of internalized gentamicin to the Golgi complex. We believe this pathway may be responsible for the early negative effects seen on protein synthesis in renal proximal epithelia after aminoglycoside administration. aminoglycosides; lysosomal-fluorescence quenching; nephrotoxicity

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis is a central readout of endothelial progenitor cell functionality. However, current assays lack kinetic measurements. To address this issue, new approaches were developed to quantitatively assess in vitro endothelial colony-forming cell (ECFC) network formation in real time. Eight parameters of network structure were quantified using novel Kinetic Analysis of Vasculogenesis (KAV) software. KAV assessment of structure complexity identified two phases of network formation. This observation guided the development of additional vasculogenic readouts. A tissue cytometry approach was established to quantify the frequency and localization of dividing ECFCs. Additionally, Fiji TrackMate was used to quantify ECFC displacement and speed at the single-cell level during network formation. These novel approaches were then implemented to identify how intrauterine exposure to maternal diabetes mellitus (DM) impairs fetal ECFC vasculogenesis. Fetal ECFCs exposed to maternal DM form fewer initial network structures, which are not stable over time. Correlation analyses demonstrated that ECFC samples with greater division in branches form fewer closed network structures. Additionally, reductions in average ECFC movement over time decrease structural connectivity. Identification of these novel phenotypes utilizing the newly established methodologies provides evidence for the cellular mechanisms contributing to aberrant ECFC vasculogenesis. [ABSTRACT FROM AUTHOR]

The commercial availability of multiphoton microscope systems has nurtured the growth of intravital microscopy as a powerful technique for evaluating cell biology in the relevant context of living animals. In parallel, new fluorescent protein (FP) biosensors have become available that enable studies of the function of a wide range of proteins in living cells. Biosensor probes that exploit Förster resonance energy transfer (FRET) are among the most sensitive indicators of an array of cellular processes. However, differences between one-photon and two-photon excitation (2PE) microscopy are such that measuring FRET by 2PE in the intravital setting remains challenging. Here, we describe an approach that simplifies the use of FRET-based biosensors in intravital 2PE microscopy. Based on a systematic comparison of many different FPs, we identified the monomeric (m) FPs mTurquoise and mVenus as particularly well suited for intravital 2PE FRET studies, enabling the ratiometric measurements from linked FRET probes using a pair of experimental images collected simultaneously. The behavior of the FPs is validated by fluorescence lifetime and sensitized emission measurements of a set of FRET standards. The approach is demonstrated using a modified version of the AKAR protein kinase A biosensor, first in cells in culture, and then in hepatocytes in the liver of living mice. The approach is compatible with the most common 2PE microscope configurations and should be applicable to a variety of different FRET probes. [ABSTRACT FROM AUTHOR]

Rab10, a mammalian homolog of the yeast Sec4p protein, has previously been associated with endocytic recycling and biosynthetic membrane transport in cultured epithelia and with Glut4 translocation in adipocytes. Here, we report that Rab10 associates with primary cilia in renal epithelia in culture and in vivo. In addition, we find that Rab10 also colocalizes with exocyst proteins at the base of nascent cilia, and physically interacts with the exocyst complex, as detected with anti-Sec8 antibodies. These data suggest that membrane transport to the primary cilum may be mediated by interactions between Rab10 and an exocyst complex located at the cilium base. [ABSTRACT FROM AUTHOR]

The ability to image calcium signals at subcellular levels within the intact depolarizing heart could provide valuable information toward a more integrated understanding of cardiac function. Accordingly, a system combining two-photon excitation with laser-scanning microscopy was developed to monitor electrically evoked [Ca[sup 2+]][sub i] transients in individual cardiomyocytes within noncontracting Langendorff-perfused mouse hearts. [Ca[sup 2+]][sub i] transients were recorded at depths ≤100 µm from the epicardial surface with the fluorescent indicators rhod-2 or fura-2 in the presence of the excitation-contraction uncoupler cytochalasin D. Evoked [Ca[sup 2+]][sub i] transients were highly synchronized among neighboring cardiomyocytes. At 1 Hz, the times from 90 to 50% (t[sub 90-50%]) and from 50 to 10% (t[sub 50-10%]) of the peak [Ca[sup 2+]][sub i] were (means ± SE) 73 ± 4 and 126 ± 10 ms, respectively, and at 2Hz, 62 ± 3 and 94 ± 6 ms(n = 19, P<0.05vs. 1Hz) in rhod-2-1oaded cardiomyocytes. [Ca[sup 2+]][sub i] decay was markedly slower in fura-2-1oaded hearts (t[sub 90-50%] at I Hz, 128 ± 9 ms and at 2 Hz, 88 ± 5 ms; t[sub 50-l0%] at 1 Hz, 214 ± 18 ms and at 2 Hz, 163 ± 7 ms; n = 19, P < 0.05 vs. rhod-2). Fura-2induced deceleration of [Ca[sup 2+]][sub i] decline resulted from increased cytosolic Ca[sup 2+] buffering, because the kinetics of rhod-2 decay resembled those obtained with fura-2 after incorporation of the Ca[sup 2+] chelator BAPTA. Propagating calcium waves and [Ca[sup 2+]][sub i] amplitude alternans were readily detected in paced hearts. This approach should be of general utility to monitor the consequences of genetic and/or functional heterogeneity in cellular calcium signaling within whole mouse hearts at tissue depths that have been inaccessible to single-photon imaging. [ABSTRACT FROM AUTHOR]

Having shown rapid trafficking of aminoglycosides to the Golgi complex in cell culture, we focused on the injurious interaction that occurs when gentamicin administration is preceded by renal ischemia. Using Texas red-labeled gentamicin as a tracer, we determined that 15 min of cellular nucleotide depletion did not significantly increase subsequent uptake. However, cells previously depleted of nucleotides accumulated significantly more Texas red-labeled gentamicin within a dispersed Golgi complex. Using Ricinus communis and Lens culinaris lectins, which label specific compartments of the Golgi complex (trans-Golgi network/trans and medial/cis compartments, respectively), we determined that the medial/cis compartment dispersed after 15 min of nucleotide depletion but the trans-Golgi network/trans compartment remained unaffected. An increase in the number of cells exhibiting disrupted medial/cisGolgi morphology after repletion in physiological media containing gentamicin was also seen. In summary, the increase in nephrotoxicity seen when ischemia precedes aminoglycoside uptake may be part of a complex mechanism initially involving increased Golgi accumulation and prolonged Golgi dispersion. The Golgi complex must then endure the effects of gentamicin accumulated in larger quantities in an aberrant physiological state. [ABSTRACT FROM AUTHOR]

Discusses a study which determined the kinetics involved in the trafficking of aminoglycosides to the Golgi complex. Materials and methods; Results; Discussion.

Intravital microscopy enables direct observation of cell biology and physiology at subcellular resolution in real time in living animals. Implanted windows extend the scope of intravital microscopy to processes extending for weeks or even months, such as disease progression or tumor development. However, a question that must be addressed in such studies is whether the imaging window, like any foreign body, triggers an inflammatory response, and whether that response alters the biological process under investigation. To directly evaluate this question, we conducted large-scale intravital microscopy of the kidney of LysM-EGFP mice over time after implantation of abdominal imaging windows. These studies demonstrate that windows stimulated a variety of changes consistent with a foreign body response. Within a few days of implantation, leukocytes were recruited to the window and the region between the window and kidney where, over the next 16 days, they increased in number in an expanding volume that developed a new vascular network. These changes were accompanied by a dramatic increase in glomerular albumin permeability within 2-5 days of implantation. Similar results were obtained from mice implanted with windows coated with poly(l-lysine)-graft-polyethylene glycol (PLL-g-PEG), but not from immune-deficient mice. These studies demonstrate the importance of evaluating whether implanted windows induce an inflammatory response, and whether that response impacts the processes under evaluation in longitudinal intravital microscopy studies. NEW & NOTEWORTHY Intravital microscopy studies of LysM-EGFP mice demonstrate that abdominal imaging windows placed over the kidney stimulated a variety of changes consistent with a foreign body response. Within a day of implantation, leukocytes were recruited to the window where, over the next 16 days, they increased in number in an expanding volume that developed a new vascular network. These changes were accompanied by a dramatic increase in glomerular permeability to albumin.

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis is a central readout of endothelial progenitor cell functionality. However, current assays lack kinetic measurements. To address this issue, new approaches were developed to quantitatively assess in vitro endothelial colony-forming cell (ECFC) network formation in real time. Eight parameters of network structure were quantified using novel Kinetic Analysis of Vasculogenesis (KAV) software. KAV assessment of structure complexity identified two phases of network formation. This observation guided the development of additional vasculogenic readouts. A tissue cytometry approach was established to quantify the frequency and localization of dividing ECFCs. Additionally, Fiji TrackMate was used to quantify ECFC displacement and speed at the single-cell level during network formation. These novel approaches were then implemented to identify how intrauterine exposure to maternal diabetes mellitus (DM) impairs fetal ECFC vasculogenesis. Fetal ECFCs exposed to maternal DM form fewer initial network structures, which are not stable over time. Correlation analyses demonstrated that ECFC samples with greater division in branches form fewer closed network structures. Additionally, reductions in average ECFC movement over time decrease structural connectivity. Identification of these novel phenotypes utilizing the newly established methodologies provides evidence for the cellular mechanisms contributing to aberrant ECFC vasculogenesis., (Copyright © 2017 the American Physiological Society.)

The commercial availability of multiphoton microscope systems has nurtured the growth of intravital microscopy as a powerful technique for evaluating cell biology in the relevant context of living animals. In parallel, new fluorescent protein (FP) biosensors have become available that enable studies of the function of a wide range of proteins in living cells. Biosensor probes that exploit Förster resonance energy transfer (FRET) are among the most sensitive indicators of an array of cellular processes. However, differences between one-photon and two-photon excitation (2PE) microscopy are such that measuring FRET by 2PE in the intravital setting remains challenging. Here, we describe an approach that simplifies the use of FRET-based biosensors in intravital 2PE microscopy. Based on a systematic comparison of many different FPs, we identified the monomeric (m) FPs mTurquoise and mVenus as particularly well suited for intravital 2PE FRET studies, enabling the ratiometric measurements from linked FRET probes using a pair of experimental images collected simultaneously. The behavior of the FPs is validated by fluorescence lifetime and sensitized emission measurements of a set of FRET standards. The approach is demonstrated using a modified version of the AKAR protein kinase A biosensor, first in cells in culture, and then in hepatocytes in the liver of living mice. The approach is compatible with the most common 2PE microscope configurations and should be applicable to a variety of different FRET probes., (Copyright © 2015 the American Physiological Society.)

Clinical studies indicate that hepatic drug transport may be altered in chronic kidney disease (CKD). Uremic solutes associated with CKD have been found to alter the expression and/or activity of hepatocyte transporters in experimental animals and in cultured cells. However, given the complexity and adaptability of hepatic transport, it is not clear whether these changes translate into significant alterations in hepatic transport in vivo. To directly measure the effect of CKD on hepatocyte transport in vivo, we conducted quantitative intravital microscopy of transport of the fluorescent organic anion fluorescein in the livers of rats following 5/6th nephrectomy, an established model of CKD. Our quantitative analysis of fluorescein transport showed that the rate of hepatocyte uptake was reduced by ∼20% in 5/6th nephrectomized rats, consistent with previous observations of Oatp downregulation. However, the overall rate of transport into bile canaliculi was unaffected, suggesting compensatory changes in Mrp2-mediated secretion. Our study suggests that uremia resulting from 5/6th nephrectomy does not significantly impact the overall hepatic clearance of an Oatp substrate., (Copyright © 2014 the American Physiological Society.)

Fluorescence microscopy is one of the most powerful tools for elucidating the cellular functions of proteins and other molecules. In many cases, the function of a molecule can be inferred from its association with specific intracellular compartments or molecular complexes, which is typically determined by comparing the distribution of a fluorescently labeled version of the molecule with that of a second, complementarily labeled probe. Although arguably the most common application of fluorescence microscopy in biomedical research, studies evaluating the "colocalization" of two probes are seldom quantified, despite a diversity of image analysis tools that have been specifically developed for that purpose. Here we provide a guide to analyzing colocalization in cell biology studies, emphasizing practical application of quantitative tools that are now widely available in commercial and free image analysis software.

Having shown rapid trafficking of aminoglycosides to the Golgi complex in cell culture, we focused on the injurious interaction that occurs when gentamicin administration is preceded by renal ischemia. Using Texas red-labeled gentamicin as a tracer, we determined that 15 min of cellular nucleotide depletion did not significantly increase subsequent uptake. However, cells previously depleted of nucleotides accumulated significantly more Texas red-labeled gentamicin within a dispersed Golgi complex. Using Ricinus communis and Lens culinaris lectins, which label specific compartments of the Golgi complex (trans-Golgi network/trans and medial/cis compartments, respectively), we determined that the medial/cis compartment dispersed after 15 min of nucleotide depletion but the trans-Golgi network/trans compartment remained unaffected. An increase in the number of cells exhibiting disrupted medial/cis-Golgi morphology after repletion in physiological media containing gentamicin was also seen. In summary, the increase in nephrotoxicity seen when ischemia precedes aminoglycoside uptake may be part of a complex mechanism initially involving increased Golgi accumulation and prolonged Golgi dispersion. The Golgi complex must then endure the effects of gentamicin accumulated in larger quantities in an aberrant physiological state.