Cortical Radial Arteries Research Articles

Super-resolution ultrasound imaging -- creating 3D in vivo images of the renal microvasculature

Aim: Our overall aim is to create 3D in vivo microvascular imaging of the renal vasculature in healthy and diabetic rats in real time. Methods-1: Super-resolution ultrasound imaging (SRUS) using intravascular microbubbles (MBs) can generate in vivo images of the renal microcirculation. Spatially separated MBs are followed over numerous image frames to create anatomical vascular images showing MB direction and velocity. Presently, images with a resolution of 50 μm are obtainable. Results-1: As diabetes mellitus is associated with micro- and macrovascular diseases also affecting the kidneys we used SRUS on Zucker Diabetic Fat rats (ZDF) aged 12-, 20- and 40-weeks. We show a significant decrease in vascular density in the cortex at week 12 before onset of proteinuria when compared to age-matched lean Zucker rats. At week 20 vascular density decreased in the outer medulla and inner medulla. No significant changes were found in vascular tortuosity. However, the SRUS images are collected over several minutes, and are challenged by tissue motion and the lack of real-time imaging. The fragile MBs limit emission pressure and thus scan depth. Hypothesis: We hypothesize that tracking erythrocytes instead of MBs will reduce these obstacles and make the method non-invasive. Resolution and scan depth will increase and scan time will shorten. This will allow an easy transition into the clinic. Results-2: Using this innovative method, we have generated images of healthy rat kidneys. Within 24 seconds, we can create an image of the renal vasculature showing the vasa recta (the capillary network penetrating the medulla) and the arcuate and cortical radial arteries. μCT scans have verified the structure of the vascular tree. However, even at these short scan times, there is considerable tissue motion from heart-beat, respiration etc. moving the image out of plane. To clearly image the true microcirculation, 3D imaging is needed. We have recently obtained this amazing step and made a 3D in vivo scan of a healthy rat kidney. The project is funded by an ERC Synergy Grant SURE, project no. 854796. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Kv channel subunits that contribute to voltage-gated K+ current in renal vascular smooth muscle.

The rat renal arterial vasculature displays differences in K(+) channel current phenotypes along its length. Small arcuate to cortical radial arteries express a delayed rectifier phenotype, while the predominant Kv current in larger arcuate and interlobar arteries is composed of both transient and sustained components. We sought to determine whether Kvalpha subunits in the rat renal interlobar and arcuate arteries form heterotetramers, which may account for the unique currents, and whether modulatory Kvbeta subunits are present in renal vascular smooth muscle cells. RT-PCR indicated the presence of several different Kvalpha subunit isoform transcripts. Co-immunoprecipitation with immunoblotting and immunohistochemical evidence suggests that a portion of the K(+) current phenotype is a heteromultimer containing delayed-rectifier Kv1.2 and A-type Kv1.4 channel subunits. RT-PCR and immunoblot analyses also demonstrated the presence of both Kvbeta1.2 and Kvbeta1.3 in renal arteries. These results suggest that heteromultimeric formation of Kvalpha subunits and the presence of modulatory Kvbeta subunits are important factors in mediating Kv currents in the renal microvasculature and suggest a potentially critical role for these channel subunits in blood pressure regulation.

Magnitude of TGF-initiated nephron-nephron interactions is increased in SHR.

We compared the tubuloglomerular feedback (TGF)-initiated nephron-nephron interaction in spontaneously hypertensive rats (SHR) and normotensive Sprague-Dawley (SD) rats. Interaction strength was assessed by measuring stop-flow pressure (delta SFP) responses in pairs of nephrons, where only one nephron of the pair was microperfused. The vascular connection was determined from casts of the nephrons and vessels; length of arteriolar separation between the two glomeruli was measured on the casts. When microperfusion rate was increased from 5 to 50 nl/min, delta SFP in perfused nephrons was 10.6 +/- 0.6 and 10.2 +/- 0.7 mmHg [not significant (NS)] in SD and SHR, respectively. In the matched unperfused nephrons from the same cortical radial artery, delta SFP was 1.3 +/- 0.2 and 2.9 +/- 0.7 mmHg (P < 0.05) in SD and SHR. When the perfused and unperfused nephron originated from different cortical radial arteries, delta SFP in the unperfused nephrons was -0.1 +/- 0.2 and 0.0 +/- 0.3 mmHg (NS) in SD and SHR, respectively. In both strains, interaction strength varied inversely with glomerular separation. When the dependence on distance was taken into account, interaction strength was about threefold greater in SHR than in SD. We conclude that the nephron-nephron interaction is significantly greater in SHR. The dependence of interaction strength on distance separating the glomeruli suggests that the interaction is propagated along the preglomerular vasculature.

Branching points of renal resistance arteries are enriched in L-type calcium channels and initiate vasoconstriction.

The morphologic structures responsible for the drop in blood pressure along the preglomerular vasculature are not completely defined. Theoretical and videomicroscopic analyses of nonrenal vascular beds implicate bifurcations of resistance arteries as important sites of hemodynamic regulation. These structures contain pacemaker cells sensitive to calcium channel blockers and appear to initiate vasomotion. In the present study, we examined the possibility of functional diversity of smooth muscle cells along resistance arteries with regard to the density of voltage-gated L-type calcium channels. Staining of microdissected renal resistance arteries with Bodipy-labeled dihydropyridine and analysis by confocal microscopy showed enhanced binding at branching points compared with the distal sites in daughter vessels. Antibodies directed against the alpha 1-subunit of the dihydropyridine-sensitive calcium channels confirmed the enhanced expression of L-type channels predominantly at the sites of bifurcations of renal resistance arteries. Fluorescence digital-image analysis of freshly microdissected branches of cortical radial (interlobular) and arcuate arteries intravitally labeled with a calcium indicator, fluo 3, identified branching points as initiator sites of depolarization-induced intracellular Ca2+ concentration ([Ca2+]i) transients, which propagated along the vascular wall at the rate of 2.0 +/- 0.7 micron/s. Videomicroscopy of blood-perfused rat juxtamedullary resistance arteries showed that branching points exhibit more pronounced contractile responses to KCl-induced depolarization than distal sites along the daughter vessels. Collectively, these results demonstrate that branching points are enriched in L-type calcium channels, a finding that suggests these structures may serve as important regulators of renal hemodynamics.

The rat renal nerves during development.

The prenatal and postnatal development of the innervation of the rat kidney has been investigated using immunocytochemical methods. The efferent innervation was studied using dopamine-beta-hydroxylase and neuropeptide Y antibodies. Calcitonin gene related peptide and substance P antibodies were used to investigate the afferent innervation. Kidneys from embryos of 14 to 20 days, from newborn rats, and from animals of 4, 10, 12, 21, 38, 60, and 90 days of age were studied. Slices of whole kidneys were analyzed, and frozen sections were used to investigate the location of the nerves in more detail. Both afferent and efferent nerves are observed inside the kidney by embryonic day 16. At birth, the afferent nerves are found (1) forming a rich plexus in the renal pelvis; (2) associated with the renal vasculature as far as the interlobular arteries (cortical radial arteries) and (3) in the corticomedullary connective tissue. The efferent innervation appears, at birth, to extend to the interlobular arteries and to the afferent arterioles of the perihilar juxtamedullary nephrons. The efferent innervation increases rapidly during the following days, and by postnatal day 21 a distribution of the innervation similar to that of the adult is observed. While the afferent innervation reaches the major target regions of the kidney by birth, the efferent does most of its expansion into the kidney postnatally. Afferent and efferent fibers are found, extrarenally and intrarenally, in the same nerve bundles. This proximity between afferent and efferent fibers may represent anatomical bases for their interaction in the adult as well as during development.

Direct visualization of renin-cell distribution in preglomerular vascular trees dissected from rat kidney.

Three methods to visualize directly the distribution of granulated renin-positive cells in vascular trees microdissected from rat kidney were developed. Kidneys were removed from anesthesized rats, hemisectioned, macerated in HCl, and soaked in distilled water for 24-48 h. Cortical preglomerular vascular trees consisting of arcuate and cortical radial arteries and afferent arterioles were microdissected with the aid of a stereomicroscope. Granulated cells can be visualized in three ways. First, under transmitted or incident light observation, granulated cells are readily distinguished from the surrounding smooth muscle cells, because of marked differences in the refractive properties of these two cell types. Second, quinacrine, a fluorescent, intravital stain selective for dense-core granules, can be administered (2 mg/kg iv) to the rat 1 h before nephrectomy. When illuminated with 440-nm light, granulated cells fluorescence strongly at 510 nm. Third, specific immunostaining for renin can be obtained with a polyclonal anti-rat renin antibody and avidin-biotin immunoperoxidase staining in vascular trees subjected to cell permeabilization with Triton. These new techniques permit the direct visualization of the distribution of granulated renin-positive cells in preglomerular vessels under conditions in which the vascular architecture is largely preserved.

Factors affecting renal microvascular blood flow in rat hyperdynamic bacteremia.

To determine whether angiotensin II and alpha-adrenergic activity contribute to the mechanism of impaired renal microvascular blood flow during hyperdynamic live Escherichia coli (E. coli) bacteremia, we used in vivo video microscopy in the chronic unilateral hydronephrotic kidney of decerebrate male Sprague-Dawley rats. Intravenous infusion of E. coli caused arteriolar constriction to 83 +/- 4% of baseline (BL) in cortical radial arteries (CRA), 82 +/- 3% of BL in afferent (AFF) arterioles, and decreased flow to 54 +/- 9% of BL. Subsequent local inhibition of renal prostaglandin synthesis with mefenamate increased preglomerular arteriolar constriction to 55 +/- 6% of BL in CRA and 51 +/- 6% of BL in AFF arterioles and decreased renal microvascular blood flow to 26 +/- 8% of BL values in E. coli animals but had no effect on control animals. Subsequent local renal angiotensin II receptor blockade with saralasin acetate increased renal microvascular blood flow in E. coli animals to 64 +/- 9% of BL by dilating CRA to 78 +/- 5% of BL and AFF arterioles to 89 +/- 5% of BL. Phentolamine caused further dilation of CRA to 104 +/- 7% BL and AFF arterioles to 116 +/- 109% and increased flow to 99 +/- 8% of BL. Acetylcholine increased diameters further to 110 +/- 3% of BL in CRA and 136 +/- 12% of BL in AFF arterioles. These data indicate that in our chronic hydronephrotic kidney model during E. coli bacteremia, renal microvascular tone is due to increased angiotensin II and alpha-adrenergic activity and some other, as yet, undefined factor.

Nephrotoxicity of a new cephalosporin, DQ-2556, in rats

A single intravenous administration of a new cephalosporin, DQ-2556, at 1200 mg/kg to Sprague-Dawley rats induced proximal tubular necrosis. The histological lesions were not closely correlated with renal cortical concentrations of DQ-2556. Development of renal injuries was examined histologically. The arcuate and cortical radial arteries of the kidneys were constricted immediately after the injection, but then became dilated and showed histological changes: penetration by erythrocytes, edematous thickening, and necrosis in the media. In addition to congestion in the outer medulla, the proximal convoluted and straight tubules exhibited the earliest changes 30 min after the injection, namely, enlargement and rounding of the mitochondria and swelling and irregular arrangement of the microvilli in the epithelial cells. Small necrotic foci of epithelium were observed from 4 hr. They were mainly distributed in the outer cortex and the outer stripe of the outer medulla 24 hr later. In the examination of hemodynamics, DQ-2556 significantly decreased the blood flow with increased vascular resistance in the renal artery during and after a single injection. These changes had disappeared wholly or partially 60 min after dosing commenced. Furthermore, Ca 2+ channel blockers markedly inhibited increases in the serum urea nitrogen and creatinine concentrations and development of the tubular necrosis and the lesions of the arterial walls, which were induced by DQ-2556. These results suggest a possible contribution of the constriction of renal artery to the tubular necrosis.

Nephrotoxicity of a New Cephalosporin, DQ-2556, in Rats

A single intravenous administration of a new cephalosporin, DQ-2556, at 1200 mg/kg to Sprague-Dawley rats induced proximal tubular necrosis. The histological lesions were not closely correlated with renal cortical concentrations of DQ-2556. Development of renal injuries was examined histologically. The arcuate and cortical radial arteries of the kidneys were constricted immediately alter the injection, but then became dilated and showed histological changes: penetration by erythrocytes, edematous thickening, and necrosis in the media. In addition to congestion in the outer medulla, the proximal convoluted and straight tubules exhibited the earliest changes 30 mm after the injection, namely, enlargement and rounding of the mitochondria and swelling and irregular arrangement of the microvilli in the epithelial cells. Small necrotic foci of epithelium were observed from 4 hr. They were mainly distributed in the outer cortex and the outer stripe of the outer medulla 24 hr later. In the examination of hemodynamics, DQ-2556 significantly decreased the blood flow with increased vascular resistance in the renal artery during and after a single injection. These changes had disappeared wholly or partially 60 mm after dosing commenced. Fur thermore, Ca2+ channel blockers markedly inhibited increases in the serum urea nitrogen and creatinine concentrations and development of the tubular necrosis and the lesions of the arterial walls, which were induced by DQ-2556. These results suggest a possible contribution of the constriction of renal artery to the tubular necrosis.

The noradrenergic innervation of the kidney in old Wistar rats

Age-related changes of the sympathetic (noradrenergic) innervation of the kidney were studied in young (3 months) and old (25 months) female Wistar rats by means' of high-pressure liquid chromatography (HPLC) with electrochemical detection and catecholamine histofluorescence techniques. Body and kidney weights were significantly increased in old rats. Renal noradrenaline levels were slightly but not significantly decreased in aged animals. The density of fluorescent catecholaminergic nerve fibres was significantly decreased in old rats, primarily within branches of renal artery (at the hilum) and within interlobular and cortical radial arteries. However, in old rats the number of axonal varicosities corresponding to the site of neurotransmitter release was slightly increased in interlobular and cortical radial arteries but not in hilar branches of the renal artery. The number of cortical tubules supplied with a catecholaminergic innervation remarkably decreased with age. These findings are indicative of age-dependent changes of the intrarenal noradrenergic innervation in Wistar rats. The lack of correspondence between the results obtained using HPLC and catecholamine histofluorescence techniques underscore the importance of evaluating the age-related changes of autonomic innervation using more than one methodology.

Autoradiographic localization of dopamine DA‐1 receptors in the rat renal vasculature using [3H]‐SCH 23390 as a ligand

1. The localization of the selective dopamine (DA) DA-1 receptor antagonist [3H]-SCH 23390 was studied in sections of rat kidney using combined radioreceptor binding and autoradiographic techniques to identify the target of DA-1 receptors in renal vasculature. 2. Binding data were consistent with the labelling by [3H]-SCH 23390 of a single population of high affinity DA-1 sites. The apparent dissociation constant value (Kd) was 4.2 nM and the maximum binding capacity value (Bmax) was 140.6 +/- 6.5 fmol mg-1 tissue. Selective DA-1 or mixed DA-1 and DA-2 receptor antagonists or agonists were able to antagonize dose-dependently [3H]-SCH 23390 binding to sections of rat kidney. DA-2 receptor selective antagonists were ineffective. 3. Light microscope autoradiography using sections exposed to nuclear emulsion for 2 weeks revealed development of specific silver grains only within proximal cortical tubules. Longer exposure times (10 weeks) caused the development of specific silver grains in proximal and distal cortical tubules and in the medial layer of intrarenal arteries. 4. Quantitative analysis of the density of silver grains within the cortical tubules and the medial layer of intrarenal arteries revealed a significantly higher density of binding sites in proximal cortical tubules than in the vasculature or in distal cortical tubules. No significant differences in the density of silver grains in the medial layer of interlobar arcuate or cortical radial arteries were observed. 5. These results suggest that, using adequate ligand concentrations and exposure times for autoradiography, both tubular and vascular DA-1 receptors can be visualized in the rat kidney. The observation of a higher density of proximal tubules DA-1 receptors is consistent with the hypothesis of a more important role of DA-1 receptors in the control of natriuresis than of renal haemodynamics.

Immunolocalization of ecto-5′-nucleotidase in the kidney by a monoclonal antibody

A monoclonal antibody IgG, has been raised against ecto-5'-nucleotidase purified from rat kidney homogenate. The specificity of the antibody was verified by immunoprecipitation. The distribution of the corresponding antigen in the rat kidney was studied by immunocytochemistry (FITC and PAP technique) in 1 micron thick cryostat sections. The antibody reacted with the brush border of proximal tubules, the apical cell membrane and the apical cytoplasm of intercalated cells in connecting tubules and collecting ducts and with interstitial cells of the cortex. Among the interstitial cells exclusively stellate shaped fibroblasts were reactive whereas rounded interstitial cells (type II interstitial cells) as well as pericytes and endothelial cells of peritubular capillaries were unreactive. Compared to the staining intensity of the fibroblasts in the cortical labyrinth the reactivity of the fibroblasts in the medullary rays of the cortex was weak or absent. Interstitial cells of the entire medulla were unreactive. Concerning the fibroblasts in the periarterial connective tissue, those surrounding the larger arteries (arcuate arteries, cortical radial arteries) were negative, those alongside afferent and efferent arterioles were positive. Endothelia of lymphatic capillaries travelling within the periarterial connective tissue were also positive. All components of the juxtaglomerular apparatus were negative. The findings are consistent with an interstitial production of adenosine, available extracellularly and thus being able to reach the major target sites of adenosine, the smooth muscles of glomerular arterioles, including the granular cells at the glomerular vascular pole.

TGF-initiated vascular interactions between adjacent nephrons in the rat kidney

We sought to determine whether tubuloglomerular feedback (TGF), activated from one nephron, affects other arterioles derived from the same cortical radial artery. Surface nephrons supplied by a single cortical radial artery were identified by injecting Ringer solution containing Fast Green from a narrow-gauge polyethylene catheter inserted via a lumbar artery into a renal artery. Stop-flow pressure was measured in an identified nephron from such a grouping. In one series, increasing end-proximal flow rate from 0 to 50 nl/min of synthetic tubular fluid in one member of an identified pair of nephrons reduced stop-flow pressure by 1.3 +/- 0.2 mmHg in the other member. When the nephrons were derived from different cortical radial arteries, the stop-flow pressure changed -0.2 +/- 0.1 mmHg. In another series, increasing flow in the adjacent nephron from 0 to 50 nl/min decreased stop-flow pressure 3.9 +/- 0.9 mmHg, and increasing flow in the adjacent nephron by the same amount when flow in the first nephron was 50 nl/min decreased stop-flow pressure 3.4 +/- 0.7 mmHg. These results indicate the operation of an interaction among nephrons derived from a common cortical radial artery. Such an interaction could produce a cooperative effect larger than that predicted from measured single-nephron responses when systemic arterial pressure changes.

Granular juxtaglomerular cells and prorenin synthesis in mice treated with enalapril

The short-term and long-term effects (for up to 98 days) of the angiotensin converting enzyme inhibitor enalapril were investigated in male and female BALB/c mice. In control animals, separate antisera to renin and its prosequence produced an identical pattern of staining in granular cells of the juxtaglomerular apparatus (JGA) a short distance from the glomerulus. After 1 day of the enalapril treatment there was a decrease in the number of JGA granular cells immunostained with antisera to both renin and its prosequence. Electron microscopy revealed degranulation of mature granules from JGA granular cells. Fusion of granules with the cell membrane was not observed, but numerous membrane-like structures (myelin figures) were identified in the cytoplasm and extracellular space, indicating possible secretion. In addition, the volume proportion of granulated cells in relation to the glomerular volume was decreased, as was renal renin content. With continuing enalapril treatment, separate antisera to renin and its prosequence stained the same granulated JGA cells with equal intensity. The cells so stained increased in number, extending down the wall of the afferent arteriole to cortical radial arteries (interlobular arteries) upstream from the glomerulus. Ultrastructural studies revealed a progressive development of cytoplasmic granulation in JGA granular cells and in smooth muscle cells extending into cortical radial arteries. Furthermore, the volume proportion of granulated cells in relation to the glomerular volume was significantly increased, as was renal renin content. Thus, short-term enalapril treatment in mice provoked rapid secretion of renin via degranulation of mature granules from JGA granular cells. In contrast, long-term enalapril treatment produced a continuing stimulus for renin synthesis, secretion and storage, resulting in an increased thickness of the afferent arteriolar wall. The mechanism for this change appears to be hypertrophy and hypergranulation of granular JGA cells and neogranulation of smooth muscle cells upstream from the glomerulus. Identification of the intrarenal mediators that induce these phenotypic changes presents an interesting challenge.