Direct evaluation of myocardial viability and stem cell engraftment demonstrates salvage of the injured myocardium.

RATIONALE: The mechanism of functional restoration by stem cell therapy remains poorly understood. Novel manganese-enhanced MRI and bioluminescence reporter gene imaging were applied to follow myocardial viability and cell engraftment, respectively. Human-placenta-derived amniotic mesenchymal stem cells (AMCs) demonstrate unique immunoregulatory and precardiac properties. In this study, the restorative effects of 3 AMC-derived subpopulations were examined in a murine myocardial injury model: (1) unselected AMCs, (2) ckit(+)AMCs, and (3) AMC-derived induced pluripotent stem cells (MiPSCs). OBJECTIVE: To determine the differential restorative effects of the AMC-derived subpopulations in the murine myocardial injury model using multimodality imaging. METHODS AND RESULTS: SCID (severe combined immunodeficiency) mice underwent left anterior descending artery ligation and were divided into 4 treatment arms: (1) normal saline control (n=14), (2) unselected AMCs (n=10), (3) ckit(+)AMCs (n=13), and (4) MiPSCs (n=11). Cardiac MRI assessed myocardial viability and left ventricular function, whereas bioluminescence imaging assessed stem cell engraftment during a 4-week period. Immunohistological labeling and reverse transcriptase polymerase chain reaction of the explanted myocardium were performed. The unselected AMC and ckit(+)AMC-treated mice demonstrated transient left ventricular functional improvement. However, the MiPSCs exhibited a significantly greater increase in left ventricular function compared with all the other groups during the entire 4-week period. Left ventricular functional improvement correlated with increased myocardial viability and sustained stem cell engraftment. The MiPSC-treated animals lacked any evidence of de novo cardiac differentiation. CONCLUSION: The functional restoration seen in MiPSCs was characterized by increased myocardial viability and sustained engraftment without de novo cardiac differentiation, indicating salvage of the injured myocardium.

Telmisartan in the diabetic murine model of acute myocardial infarction: dual contrast manganese-enhanced and delayed enhancement MRI evaluation of the peri-infarct region.

BACKGROUND: A 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 RESULTS: Dual 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). CONCLUSION: The 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.

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.

The role of TRPC6 calcium channels and P2 purinergic receptors in podocyte mechanical and metabolic sensing.

Podocyte calcium (Ca2+) signaling plays important roles in the (patho)physiology of the glomerular filtration barrier. Overactivation of podocyte transient receptor potential canonical (TRPC) channels including TRPC6 and purinergic signaling via P2 receptors that are known mechanosensors can increase podocyte intracellular Ca2+ levels ([Ca2+]i) and cause cell injury, proteinuria and glomerular disease including in diabetes. However, important mechanistic details of the trigger and activation of these pathways in vivo in the intact glomerular environment are lacking. Here we show direct visual evidence that podocytes can sense mechanical overload (increased glomerular capillary pressure) and metabolic alterations (increased plasma glucose) via TRPC6 and purinergic receptors including P2Y2. Multiphoton microscopy of podocyte [Ca2+]i was performed in vivo using wild-type and TRPC6 or P2Y2 knockout (KO) mice expressing the calcium reporter GCaMP3/5 only in podocytes and in vitro using freshly dissected microperfused glomeruli. Single-nephron intra-glomerular capillary pressure elevations induced by obstructing the efferent arteriole lumen with laser-induced microthrombus in vivo and by a micropipette in vitro triggered >2-fold increases in podocyte [Ca2+]i. These responses were blocked in TRPC6 and P2Y2 KO mice. Acute elevations of plasma glucose caused >4-fold increases in podocyte [Ca2+]i that were abolished by pharmacological inhibition of TRPC6 or P2 receptors using SAR7334 or suramin treatment, respectively. This study established the role of Ca2+ signaling via TRPC6 channels and P2 receptors in mechanical and metabolic sensing of podocytes in vivo, which are promising therapeutic targets in conditions with high intra-glomerular capillary pressure and plasma glucose, such as diabetic and hypertensive nephropathy.

Activation of the succinate receptor GPR91 in macula densa cells causes renin release.

Macula densa (MD) cells of the juxtaglomerular apparatus (JGA) are salt sensors and generate paracrine signals that control renal blood flow, glomerular filtration, and release of the prohypertensive hormone renin. We hypothesized that the recently identified succinate receptor GPR91 is present in MD cells and regulates renin release. Using immunohistochemistry, we identified GPR91 in the apical plasma membrane of MD cells. Treatment of MD cells with succinate activated mitogen-activated protein kinases (MAPKs; p38 and extracellular signal-regulated kinases 1/2) and cyclooxygenase 2 (COX-2) and induced the synthesis and release of prostaglandin E(2), a potent vasodilator and classic paracrine mediator of renin release. Using microperfused JGA and real-time confocal fluorescence imaging of quinacrine-labeled renin granules, we detected significant renin release in response to tubular succinate (EC(50) 350 microM). Genetic deletion of GPR91 (GPR91(-/-) mice) or pharmacologic inhibition of MAPK or COX-2 blocked succinate-induced renin release. Streptozotocin-induced diabetes caused GPR91-dependent upregulation of renal cortical phospho-p38, extracellular signal-regulated kinases 1/2, COX-2, and renin content. Salt depletion for 1 wk increased plasma renin activity seven-fold in wild-type mice but only 3.4-fold in GPR91(-/-) mice. In summary, MD cells can sense alterations in local tissue metabolism via accumulation of tubular succinate and GPR91 signaling, which involves the activation of MAPKs, COX-2, and the release of prostaglandin E(2). This mechanism may be integral in the regulation of renin release and activation of the renin-angiotensin system in health and disease.

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.

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.

Comparison of elimination and cardiovascular effects of adenine nucleosides administered intrapericardially or intravenously in anesthetized dog.

Intrapericardial (IP) administration of certain cardioactive agents allows investigation of local pharmacological actions on the heart and may carry potential benefit to influence myocardial function. The cardioprotective adenosine (ADO) and inosine (INO) may be the most representative candidates. Elimination and cardiovascular effects of IP and intravenously (IV) applied ADO and INO were compared on anesthetized dogs. Their pericardial and systemic concentrations were measured after consecutive administration of increasing ADO and INO doses. In the case of IP administration at the end of the incubation period, pericardial concentrations of adenine nucleosides significantly exceeded the control values. However, the IV applied ADO and INO were rapidly metabolized in the systemic plasma. As characteristic hemodynamic effects, small but sustained decrease in heart rate (IP ADO) and increase in myocardial contractility (IP INO) were observed. During IV administration, ADO and INO exerted remarkable effects on all hemodynamic variables, which then gradually disappeared in 15 minutes. In summary, the elimination of ADO and INO was significantly slower in the pericardial fluid than in the plasma. Considering the balanced cardiac actions and lack of strong systemic hemodynamic effects, IP administration of adenine nucleosides may suggest a promising approach in the local treatment of the diseased heart.

Imaging the renin-angiotensin system: an important target of anti-hypertensive therapy.

Multiphoton fluorescence microscopy allows visualization, manipulation, and quantification of the structure-function relationships between pharmacological interventions and their physiological effects. The application of these methods to live animals permits direct observation of acute physical responses that lack chemically detectable signals in the blood or urine and would otherwise remain unknown. With the use of special fluorescent dyes, chemical/hormonal responses may also be detected. The delivery and site-specific effects of drugs can be monitored in real-time. The capacity to simultaneously visualize both proximal and distal segments of the nephron permits observation of the dynamic processes within the living kidney and a quantitative assessment of the various operations. Consequently, a clinically valuable and pending application for multi-photon microscopy will be to provide real-time, quantitative imaging of basic organ functions and their responses to therapeutic intervention. Imaging of the intra-renal renin content and enzymatic activity of renin in situ and in real-time is a new, more informative measure of RAS activity. Direct visualization of the molecular and cellular components of renin release signals and the interactions between the vascular endothelium, tubular epithelium, local mediators, and the renin producing cells provides great insight for drug development. Examples of how the effects of various RAS inhibitors can be visualized in the intact kidney are provided: including angiotensin converting enzyme inhibition (captopril), angiotensin II type 1 receptor blockade (olmesartan), and renin inhibition (aliskiren). The site-specific actions of diuretics, like furosemide, have also been visualized. Quantitative imaging of basic renal functions in health and disease can provide key information to assess the delivery and effects of pharmaceutical interventions.

Intrapericardial angiotensin II stimulates endothelin-1 and atrial natriuretic peptide formation of the in situ dog heart.

Angiotensin (AT) II, endothelin (ET)-1, and atrial natriuretic peptide (ANP) play an important role in cardiovascular regulatory processes under physiologic and pathophysiologic conditions. All of these agents are present in the pericardial fluid, and alteration of their pericardial concentrations mirror changes in the myocardial interstitium. Moreover, the composition the pericardial fluid may also reflect the myocardial interaction of these agents. The local myocardial effects of AT II on cardiac ET-1 and ANP production, as well as on cardiovascular function, was studied by intrapericardial (ip) administration of AT II (0.125-1.0 microg/kg) to the in situ dog heart (n = 8). Big ET, ET-1, and ANP [1-28] fragment concentrations were measured by enzyme-linked immunosorbent assay in pericardial infusate samples and in peripheral blood before and after an AT II treatment of 15 mins. Systemic blood pressure (BP), heart rate (HR), and left ventricular contractility (dP/dt) were also recorded. In our studies, the pericardial big ET (but not ET-1) concentration was increased to a maximum value of 139 +/- 28 versus 74 +/- 12 pg/ml (control; P < 0.02) with ip AT II administration, with parallel elevations of the pericardial ANP levels (36.8 +/- 7.2 vs. 24.4 +/- 3.6 ng/ml; P < 0.05). The ip administration of AT II did not influence HR, and it elicited moderate changes in BP (BP(max), +14 +/- 2 mm Hg, P < 0.001; dP/dt(max), +10 +/- 3%, P < 0.02). The plasma levels of big ET, ET-1, and ANP did not change significantly. The results suggest that AT II promotes production of big ET and ANP in the heart. However, no detectable conversion of big ET-1 to ET-1 was observed within 15 mins. The myocardial formation of big ET-1 and ANP occurred, at least in part, independently of the changes in cardiovascular function.

Imaging renin content and release in the living kidney.

Renin release is the first, and at least initially, the rate-limiting step in the activation of the renin-angiotensin system, which helps to maintain body salt and water balance. Recent advances in our understanding of pathophysiology have generated a renewed interest in the multiple roles of renin and prorenin as a hormone, enzyme, and signaling molecule. The assays available to measure renin content, release and tissue activity are complex, indirect and work with significant internal errors. We developed an imaging approach to directly visualize renin content and study the dynamics of both the release and tissue activity of renin. Our experimental model uses multiphoton fluorescence microscopy, which is ideal for deep optical sectioning of the living renal tissue. Here we review the application of this renin imaging approach to the dissected, in vitro microperfused glomerulus as well as in the intact kidney in vivo.

Intrapericardial adenine nucleosides induce elevation of endothelin-1 concentration in the pericardial space of the dog heart.

The adenine nucleosides, adenosine (ADO) and inosine (INO), and the endothelin-1 (ET-1) play an important role in the regulation of coronary and myocardial functions. The pericardial fluid accumulates these regulatory agents and reflects well their levels in the myocardial interstitium. This offers a possibility to examine the local adenine nucleoside-endothelin interactions in heart tissue. We have previously demonstrated that increased levels of intrapericardial (i.p.) ET-1 enhanced significantly the adenine nucleoside concentrations of the pericardial fluid samples. In this study the effects of i.p.-administered ADO and INO were investigated on the pericardial concentrations of ET-1 in the in situ dog heart. The ET-1 concentrations were determined (enzymelinked immunosorbent assay) in the samples obtained from the pericardial fluid and from the arterial plasma before and after i.p. administration of increasing doses of ADO (10, 100 and 1000 muM, n = 8) and INO (1, 10 and 100 mM, n = 8). Standard hemodynamic variables were recorded continuously. We found that i.p. ADO and INO induced dose-dependent increases of pericardial ET-1 levels after a 15-minute incubation period in pericardial (ET-1max,ADO, +121 +/- 26%, P < 0.02; and ET-1max,INO, +84 +/- 27%, P < 0.05) but not in the plasma samples. The i.p. nucleosides elicited slight but characteristic alterations in the heart rate and ventricular contractility (dP/dt). The results suggest that the pericardial adenine nucleosides interact with the myocardial ET-1 and this effect may be provoked from, and can also be detected in, the pericardium.

Effects of intrapericardially administered endothelin-1 on pericardial natriuretic peptide concentrations of the in situ dog heart.

It has recently been proposed that endothelin-1 (ET-1) is an important activator of natriuretic peptide [atrial natriuretic peptide (ANP) and the brain natriuretic peptide (BNP)] release in the heart and may mediate ANP and BNP deliberation to myocardial stretch and ischemia. The close inter-relationship between ET-1 and natriuretic peptide release was indicated mainly by the results of in vitro studies. In vivo, the local alterations of ANP and BNP levels in the myocardial interstitium can be well characterized by the changes of their pericardial fluid concentrations. The effect of the intrapericardially administered ET-1 on natriuretic peptide concentrations was studied on the in situ dog heart (n = 8). Control and three consecutive infusate samples were removed from the pericardial sac following ET-1 administration (150 pmol/kg) and parallel peripheral blood samples were taken to determine the ANP and BNP concentrations (enzyme-linked immunosorbent assay). Standard hemodynamic parameters were recorded continuously. In our results the intrapericardial ET-1 increased pericardial ANP but not BNP concentrations significantly (control versus ANPmax, 37 +/- 5 ng/mL versus 94 +/- 12 ng/mL; P < 0.02). ET-1 elicited significant ST elevations without changes of the hemodynamic values and the natriuretic peptide levels in the arterial plasma samples. In conclusion, intrapericardial ET-1 effectively stimulated the myocardial ANP release, which was reflected as elevation in the pericardial ANP level. The results also support the hypothesis that important regulatory mechanisms might be activated from the pericardium.

Cardiovascular responses to catecholamines and interactions with endothelin-1 and adenine nucleosides in the pericardium of the dog heart.

The pericardial fluid may accumulate endogenous regulatory agents, such as catecholamines, endothelin or adenine nucleosides. However, very little information is available on the cardiovascular effects of intrapericardial (i.p.) catecholamines and their interaction with the endogenous endothelins and adenine nucleosides. The cardiovascular effects of increasing doses of i.p.- administered dopamine boluses (0.06-8 micromol/kg, n = 8) were studied in the in situ canine heart: systemic blood pressure, heart rate and left ventricular dP/dt were recorded, and pericardial infusate samples were obtained to measure the changes in endothelin-1 (ET-1), adenosine and inosine levels (enzyme-linked immunosorbent assay and high-performance liquid chromatography methods, respectively). The responses to i.p. dopamine were compared with the effects of i.p. norepinephrine boluses (0.004-0.5 micromol/kg, n = 8). Dopamine elicited dose-dependent increases of heart rate (P < 0.01), and the highest dose of dopamine resulted in significant elevation in dP/dt and blood pressure (P < 0.01) with a nearly twofold increase of i.p. ET-1 (from 14.3 +/- 0.1 pg/mL to 26.1 +/- 0.1 pg/mL, P < 0.02) and a more than threefold augmentation of i.p. adenosine (from 2.9 +/- 0.5 microM to 11.1 +/- 3.0 microM, P < 0.05), but not of inosine levels. Similar responses were obtained with i.p. norepinephrine. The results confirm that i.p. catecholamines exert significant hemodynamic effects and modulate ET-1 and adenosine release from the heart. However, the pattern of catecholamine actions initiated from the pericardium may characteristically differ from that of intravascular ones.

Nitrogen oxide blockade does not aggravate the endothelin-1-induced myocardial ischemia and release of purine metabolites from the dog heart.

Increased intrapericardial levels of endothelin-1 (ET-1) induce myocardial ischemia and concomitant release of the purine metabolites adenosine (ADO), inosine (INO) and hypoxanthine (HXA) into the pericardial fluid. However, the potential modulatory role of nitrogen monoxide in compensating the ET-1-induced ischemic stress is not fully elucidated. The pericardial elevations of purine metabolite concentrations in the pericardial fluid after ET-1 administration (150 pmol/kg intrapericardially) were measured in the in situ dog heart with (n = 6) or without (n = 5) systemic nitrogen monoxide synthase blockade (30 mg/kg (G)-nitro-L-arginine methyl ester, followed by 6 mg/min intravenously). After control sampling, three consecutive pericardial infusate samples (ET1, ET2, ET3) were obtained for purine metabolite determinations (high-performance liquid chromatography-ultraviolet). It was found that intrapericardial ET-1 elevated the pericardial purine metabolite concentrations significantly in both groups. No significant differences were detected between the control and (G)-nitro-L-arginine methyl ester-treated groups in ischemic changes of pericardial ADOmax (+3.27 +/- 1.13 microM versus +1.84 +/- 0.56 microM), INOmax (+15.21 +/- 2.3 microM versus +12.09 +/- 4.04 microM) and HXAmax (+16.34 +/- 2.98 microM versus +17.09 +/- 5.22 microM) levels and in the maximal ST elevations (0.43 +/- 0.05 mV versus 0.61 +/- 0.08 mV). The hemodynamic variables did not change with ET-1 administration. In conclusion, systemic nitrogen monoxide synthase blockade does not aggravate the ET-1-induced acute myocardial ischemia and the release of purine metabolites, suggesting that endogenous nitrogen monoxide is not a supplementary factor to purine metabolites in this type of coronary adaptive responses.

Human amniotic mesenchymal stem cell-derived induced pluripotent stem cells may generate a universal source of cardiac cells.

Human amniotic mesenchymal stem cells (hAMSCs) demonstrated partially pluripotent characteristics with a strong expression of Oct4 and Nanog genes and immunomodulatory properties characterized by the absence of HLA-DR and the presence of HLA-G and CD59. The hAMSCs were reprogrammed into induced pluripotent stem cells (iPSCs) that generate a promising source of universal cardiac cells. The hAMSC-derived iPSCs (MiPSCs) successfully underwent robust cardiac differentiation to generate cardiomyocytes. This study investigated 3 key properties of the hAMSCs and MiPSCs: (1) the reprogramming efficiency of the partially pluripotent hAMSCs to generate MiPSCs; (2) immunomodulatory properties of the hAMSCs and MiPSCs; and (3) the cardiac differentiation potential of the MiPSCs. The characteristic iPSC colony formation was observed within 10 days after the transduction of the hAMSCs with a single integration polycistronic vector containing 4 Yamanaka factors. Immunohistology and reverse transcription-polymerase chain reaction assays revealed that the MiPSCs expressed stem cell surface markers and pluripotency-specific genes. Furthermore, the hAMSCs and MiPSCs demonstrated immunomodulatory properties enabling successful engraftment in the SVJ mice. Finally, the cardiac differentiation of MiPSCs exhibited robust spontaneous contractility, characteristic calcium transience across the membrane, a high expression of cardiac genes and mature cardiac phenotypes, and a contractile force comparable to cardiomyocytes. Our results demonstrated that the hAMSCs are reprogrammed with a high efficiency into MiPSCs, which possess pluripotent, immunomodulatory, and precardiac properties. The MiPSC-derived cardiac cells express a c-kit cell surface marker, which may be employed to purify the cardiac cell population and enable allogeneic cardiac stem cell therapy.

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.

Electrotonic vascular signal conduction and nephron synchronization.

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.

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.