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BackgroundA novel MRI technique, employing dual contrast manganese-enhanced MRI (MEMRI) and delayed enhancement MRI (DEMRI), can evaluate the physiologically unstable peri-infarct region. Dual contrast MEMRI-DEMRI enables comprehensive evaluation of telmisartan to salvage the peri-infarct injury to elucidate the underlying mechanism of restoring the ischemic cardiomyopathy in the diabetic mouse model.Methods and resultsDual contrast MEMRI-DEMRI was performed on weeks 1, 2, and 4 following initiation of telmisartan treatment in 24 left anterior descendent artery ligated diabetic mice. The MRI images were analyzed for core infarct, peri-infarct, left ventricular end-diastolic, end-systolic volumes, and the left ventricular ejection fraction (LVEF). Transmission electron microscopy (TEM) and real-time PCR were used for ex vivo analysis of the myocardium. Telmisartan vs. control groups demonstrated significantly improved LVEF at weeks 1, 2, and 4, respectively (33 ± 7 %*** vs. 19 ± 5 %, 29 ± 3 %*** vs. 22 ± 4 %, and 31 ± 2 %*** vs 18 ± 6 %, ***p < 0.001). The control group demonstrated significant differences in the scar volume measured by MEMRI and DEMRI, demonstrating peri-infarct injury. Telmisartan group significantly salvaged the peri-infarct injury. The myocardial effects were validated by TEM, which confirmed the presence of the injured but viable cardiomyocyte morphology in the peri-infarct region and by flow cytometry of venous blood, which demonstrated significantly increased circulating endothelial progenitor cells (EPCs).ConclusionThe improved cardiac function in ischemic cardiomyopathy of diabetic mice by telmisartan is attributed to the attenuation of the peri-infarct injury by the angiogenic effects of EPCs to salvage the injured cardiomyocytes. Dual-contrast MEMRI-DEMRI technique tracked the therapeutic effects of telmisartan on the injured myocardium longitudinally.

Tubuloglomerular feedback (TGF) and the myogenic mechanism control afferent arteriolar diameter in each nephron and regulate blood flow. Both mechanisms generate self-sustained oscillations, the oscillations interact, TGF modulates the frequency and amplitude of the myogenic oscillation, and the oscillations synchronize; a 5:1 frequency ratio is the most frequent. TGF oscillations synchronize in nephron pairs supplied from a common cortical radial artery, as do myogenic oscillations. We propose that electrotonic vascular signal propagation from one juxtaglomerular apparatus interacts with similar signals from other nephrons to produce synchronization. We tested this idea in tubular-vascular preparations from mice. Vascular smooth muscle cells were loaded with a fluorescent voltage-sensitive dye; fluorescence intensity was measured with confocal microscopy. Perfusion of the thick ascending limb activated TGF and depolarized afferent arteriolar smooth muscle cells. The depolarization spread to the cortical radial artery and other afferent arterioles and declined with distance from the perfused juxtaglomerular apparatus, consistent with electrotonic vascular signal propagation. With a mathematical model of two coupled nephrons, we estimated the conductance of nephron coupling by fitting simulated vessel diameters to experimental data. With this value, we simulated nephron pairs to test for synchronization. In single-nephron simulations, the frequency of the TGF oscillation varied with nephron length. Coupling nephrons of different lengths forced TGF frequencies of both pair members to converge to a common value. The myogenic oscillations also synchronized, and the synchronization between the TGF and the myogenic oscillations showed an increased stability against parameter perturbations. Electronic vascular signal propagation is a plausible mechanism for nephron synchronization. Coupling increased the stability of the various oscillations. renal autoregulation; tubuloglomerular feedback; myogenic mechanism; coupled oscillators; coupled nephrons; stability

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 fuorescence 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. gap junction; Cre/loxP technique; lacZ

Endothelial intracellular calcium ([[[Ca.sup.+]].sub.i]) plays an important role in the function of the juxtaglomerular vasculature. The present studies aimed to identify the existence and molecular elements of an endothelial calcium wave in cultured glomerular endothelial cells (GENC). GENCs on glass coverslips were loaded with Fluo-4/Fura red, and ratiometric [[[Ca.sup.+]].sub.i] imaging was performed using fluorescence confocal microscopy. Mechanical stimulation of a single GENC caused a nine-fold increase in [[[Ca.sup.+]].sub.i], which propagated from cell to cell throughout the monolayer (7.9 [+ or -] 0.3 [micro]m/s) in a regenerative manner (without decrement of amplitude, kinetics, and speed) over distances >400 [micro]m. Inhibition of voltage-dependent calcium channels with nifedipine had no effect on the above parameters, but the removal of extracellular calcium reduced [DELTA][[[Ca.sup.+]].sub.i] by 50%. Importantly, the gap junction uncoupler [alpha]-glycyrrhetinic acid or knockdown of connexin 40 (Cx40) by transfecting GENCs with Cx40 short interfering RNA (siRNA) almost completely eliminated [DELTA][[[Ca.sup.+]].sub.i] and the calcium wave. Breakdown of extracellular ATP using a scavenger cocktail (apyrase and hexokinase) or nonselective inhibition of purinergic P2 receptors with suramin, had similar blocking effects. Scraping cells off along a line eliminated physical contact between cells but did not effect calcium wave propagation. Using an ATP biosensor technique, we detected a significant elevation in extracellular ATP ([DELTA] = 76 [+ or -] 2 [micro]M) during calcium wave propagation, which was abolished by Cx40 siRNA treatment ([DELTA] = 6 [+ or -] 1 [micro]M). These studies suggest that connexin 40 hemichannels and extracellular ATP are key molecular elements of the glomerular endothelial calcium wave, which may serve important juxtaglomerular functions. purinergic receptors; gap junction; connexin hemichannels; glomerular filtration; renin release

The glomerular filtration barrier (GFB) is generally considered to consist of three layers: fenestrated glomerular endothelium, glomerular basement membrane, and filtration slits between adjacent podocyte foot processes. Detailed anatomic examination of the GFB has revealed a novel abluminal structure, the subpodocyte space (SPS), identified as the labyrinthine space between the underside of podocyte cell body/primary processes and the foot processes. The SPS covers 50-65% of the filtration surface of the GFB, indicating that SPS may influence glomerular permeability. We have examined the contribution of the SPS to the permeability characteristics of the GFB using multiphoton microscopy techniques in isolated, perfused glomeruli and in the intact kidney in vivo. SPS were identified using this technique, with comparable dimensions to SPS examined with electron microscopy. The passage of the intermediate-weight molecule rhodamine-conjugated 10-kDa dextran, but not the low-weight molecule lucifer yellow ([approximately equal to] 450 Da), accumulated in SPS-covered regions of the GFB, compared with GFB regions not covered by SPS ('naked regions'). Net lucifer yellow flux (taken to indicate fluid flux) through identifiable SPS regions was calculated to be 66-75% of that occurring through naked regions. These observations indicate both ultrafiltration and hydraulic resistance imparted by the SPS, demonstrating the potential physiological contribution of the SPS to glomerular permeability. glomerulus; permeability; podocyte; multiphoton

Earlier electron microscopy studies demonstrated morphological signs of fluid flow in the juxtaglomerular apparatus (JGA), including fenestrations of the afferent arteriole (AA) endothelium facing renin granular cells. We aimed to directly visualize fluid flow in the JGA, the putative function of the fenestrated endothelium, using intravital multiphoton microscopy of Munich-Wistar rats and C57BL6 mice. Renin content of the AA correlated strongly with the length of the fenestrated, filtering AA segment. Fluorescence of the extracellular fluid marker lucifer yellow (LY) injected into the cannulated femoral vein in bolus was followed in the renal cortex by real-time imaging. LY was detected in the interstitium around the JG AA before the plasma LY filtered into Bowman's capsule and early proximal tubule. The fluorescence intensity of LY in the JGA interstitium was 17.9 [+ or -] 3.5% of that in the AA plasma (n = 6). The JGA fluid flow was oscillatory, consisting of two components: a fast (one every 5-10 s) and a slow (one every 45-50 s) oscillation, most likely due to the rapid transmission of both the myogenic and tubuloglomerular feedback (TGF)-mediated hemodynamic changes. LY was also detected in the distal tubular lumen about 2-5 s later than in the AA, indicating the flow of JGA interstitial fluid through the macula densa. In the isolated microperfused JGA, blocking the early proximal tubule with a micropipette caused significant increases in MD cell volume by 62 [+ or -] 4% (n = 4) and induced dilation of the intercellular lateral spaces. In summary, significant and dynamic fluid flow exists in the JGA which may help filter the released renin into the renal interstitium (endocrine function). It may also modulate TGF and renin signals in the JGA (hemodynamic function). intravital microscopy; fenestration; short-loop feedback; quinacrine; lucifer yellow

Multiphoton fluorescence microscopy offers the advantages of deep optical sectioning of living tissue with minimal phototoxicity and high optical resolution. More importantly, dynamic processes and multiple functions of an intact organ can be visualized in real time using noninvasive methods, and quantified. These studies aimed to extend existing methods of multiphoton fluorescence imaging to directly observe and quantify basic physiological parameters of the kidney including glomerular filtration rate (GFR) and permeability, blood flow, urinary concentration/dilution, renin content and release, as well as more integrated and complex functions like the tubuloglomerular feedback (TGF)-mediated oscillations in glomerular filtration and tubular flow. Streptozotocin-induced diabetes significantly increased single-nephron GFR (SNGFR) from 32.4 [+ or -] 0.4 to 59.5 [+ or -] 2.5 nl/min and glomerular permeability to a 70-kDa fluorophore approximately eightfold. The loop diuretic furosemide 2-fold diluted and increased ~10-fold the volume of distal tubular fluid, while also causing the release of 20% of juxtaglomerular renin content. Significantly higher speeds of individual red blood cells were measured in intraglomerular capillaries (16.7 [+ or -] 0.4 mm/s) compared with peritubular vessels (4.7 [+ or -] 0.2 mm/s). Regular periods of glomerular contraction-relaxation were observed, resulting in oscillations of filtration and tubular flow rate. Oscillations in proximal and distal tubular flow showed similar cycle times (~45 s) to glomerular filtration, with a delay of ~5-10 and 25-30 s, respectively. These innovative technologies provide the most complex, immediate, and dynamic portrayal of renal function, clearly depicting the components and mechanisms involved in normal physiology and pathophysiology. intravital microscopy; diabetic nephropathy; single-nephron glomerular filtration rate; red blood cell velocity; quinacrine; rhodamine; lucifer yellow; proteinuria doi:10.1152/ajprenal.00521.2005

Multiphoton fluorescence microscopy offers the advantages of deep optical sectioning of living tissue with minimal phototoxicity and high optical resolution. More importantly, dynamic processes and multiple functions of an intact organ can be visualized in real time using noninvasive methods, and quantified. These studies aimed to extend existing methods of multiphoton fluorescence imaging to directly observe and quantify basic physiological parameters of the kidney including glomerular filtration rate (GFR) and permeability, blood flow, urinary concentration/dilution, renin content and release, as well as more integrated and complex functions like the tubuloglomerular feedback (TGF)-mediated oscillations in glomerular filtration and tubular flow. Streptozotocin-induced diabetes significantly increased single-nephron GFR (SNGFR) from 32.4 ± 0.4 to 59.5 ± 2.5 nl/min and glomerular permeability to a 70-kDa fluorophore approximately eightfold. The loop diuretic furosemide 2-fold diluted and increased ∼10-fold the volume of distal tubular fluid, while also causing the release of 20% of juxtaglomerular renin content. Significantly higher speeds of individual red blood cells were measured in intraglomerular capillaries (16.7 ± 0.4 mm/s) compared with peritubular vessels (4.7 ± 0.2 ram/s). Regular periods of glomerular contraction-relaxation were observed, resulting in oscillations of filtration and tubular flow rate. Oscillations in proximal and distal tubular flow showed similar cycle times (∼45 s) to glomerular filtration, with a delay of ∼5–10 and 25–30 s, respectively. These innovative technologies provide the most complex, immediate, and dynamic portrayal of renal function, clearly depicting the components and mechanisms involved in normal physiology and pathophysiology. [ABSTRACT FROM AUTHOR]

An abstract of the conference paper "In vivo detection and treatment of ischemia-induced cardiac apoptosis using an MRI-detectable molecular probe and an alpha-adrenergic receptor agonist," by Justin Lam, and colleagues is presented.

Uric acid (UA) is a metabolite of dietary fructose and cellular purine degradation and is one of the main risk factors of cardiovascular disease and hypertension. UA activates the intrarenal renin-angiotensin system (RAS), but the mechanism is not known. Renin release from the juxtaglomerular apparatus (JGA), the rate-limiting step of RAS activation, is highly regulated. We hypothesized that UA directly causes JGA renin release. Fluorescence confocal microscopy using quinacrine showed that perfusion of 100 µM UA into in vitro microperfused mouse JGA preparations caused a 36 ± 6% decrease of JGA renin content (n=4,P<0.001). UA caused no acute increases in NO production or calcium signaling in the afferent arteriole or glomerular endothelial cells, evaluated by DAF-FM and Fluo-4/Fura red imaging. Quantitative in vivo multiphoton imaging of Munich-Wistar rat kidneys assessed UA (0.1 mg/kg iv)-induced changes in kidney function. Hemodynamic changes included an acute 35% increase in single-nephron glomerular filtration rate (snGFR) from 31 ± 1 to 42 ± 2 nl/min (n=8,P<0.001). Cortical blood flow increased: peritubular capillary red blood cell velocity rose from 6 ± 1 to 13 ± 1 µm/ms (n=10,P<0.001). Blood pressure showed no significant changes. This study showed that hyperuricemia may mimic diabetic hypertension: early hyperfunctionality precedes late failure. Non-endothelial cells of the JGA, possibly JG or macula densa cells, are involved in UA-mediated renin release. [ABSTRACT FROM AUTHOR]

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., (Copyright 2008 by John Wiley & Sons, Inc.)