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.

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.

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.

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.

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.

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.

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.

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.

Connexin 30.3 is expressed in the kidney but not regulated by dietary salt or high blood pressure.

Several isoforms of connexin (Cx) proteins have been identified in a variety of tissues where they play a role in intercellular communication, either as the components of gap junctions or as large, nonselective pores known as hemichannels. This investigation seeks to identify the localization and regulation of Cx30.3 in mouse, rat, and rabbit kidney using a Cx30.3(+/lacZ) transgenic approach and immunofluorescence. Cx30.3 was detected in all three species and predominantly in the renal medulla. Both the nuclear lacZ staining indicative of Cx30.3 expression and indirect immunohistochemistry provided the same results. Cx30.3 immunolabeling was mainly punctate in the mouse, typical for gap junctions. In contrast, it showed continuous apical plasma membrane localization in certain tubule segments in the rat and rabbit kidney, suggesting that it may also function as hemichannels. In the cortex, Cx30.3 was localized in the intercalated cells of the cortical collecting duct, because the immunoreactive cells did not label for AQP2, a marker for principal cells. In the medulla, dense Cx30.3 staining was confined to the ascending thin limbs of the loop of Henle, because the immunoreactive cells did not label for AQP1, a marker of the descending thin limbs. Immunoblotting studies indicated that Cx30.3 expression was unchanged in response to either high or low salt intake or in spontaneously hypertensive rats. Cx30.3 appears to be constitutively expressed in certain renal tubular segments and cells and its role in overall kidney function remains to be investigated.

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.

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.

Connexin 40 and ATP-dependent intercellular calcium wave in renal glomerular endothelial cells.

Endothelial intracellular calcium ([Ca(2+)](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(2+)](i) imaging was performed using fluorescence confocal microscopy. Mechanical stimulation of a single GENC caused a nine-fold increase in [Ca(2+)](i), which propagated from cell to cell throughout the monolayer (7.9 +/- 0.3 microm/s) in a regenerative manner (without decrement of amplitude, kinetics, and speed) over distances >400 microm. Inhibition of voltage-dependent calcium channels with nifedipine had no effect on the above parameters, but the removal of extracellular calcium reduced Delta[Ca(2+)](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(2+)](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 +/- 2 microM) during calcium wave propagation, which was abolished by Cx40 siRNA treatment (Delta = 6 +/- 1 microM). 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.

The collecting duct is the major source of prorenin in diabetes.

In addition to the juxtaglomerular apparatus, renin is also synthesized in renal tubular epithelium, including the collecting duct (CD). Angiotensin (Ang) II differentially regulates the synthesis of juxtaglomerular (inhibition) and CD (stimulation) renin. Because diabetes mellitus, a disease with high intrarenal renin-Ang system and Ang II activity, is characterized by high prorenin levels, we hypothesized that the CD is the major source of prorenin in diabetes. Renin granular content was visualized using in vivo multiphoton microscopy of the kidney in diabetic Munich-Wistar rats. Diabetes caused a 3.5-fold increase in CD renin, in contrast to less pronounced juxtaglomerular changes. Ang II type 1 receptor blockade with Olmesartan reduced CD renin to control levels but significantly increased juxtaglomerular renin. Using a fluorogenic renin assay, the prorenin component of CD renin content was measured by assessing the difference in enzymatic activity of medullary homogenates before and after trypsin activation of prorenin. Trypsinization caused no change in control renin activity but a 5-fold increase in diabetes. Studies on a CD cell line (M1) showed a 22-fold increase in renin activity after trypsinization and a further 35-fold increase with Ang II treatment. Therefore, prorenin significantly contributes to baseline CD renin. Diabetes, possibly via Ang II, greatly stimulates CD prorenin and causes hyperplasia of renin-producing connecting segments. These novel findings suggest that, in a rat model of diabetes, prorenin content and release from the CD may be more important than the juxtaglomerular apparatus in contrast to the existing paradigm.

Evidence for restriction of fluid and solute movement across the glomerular capillary wall by the subpodocyte space.

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 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.

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.

Fluid flow in the juxtaglomerular interstitium visualized in vivo.

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 +/- 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 +/- 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).