Bentley, Michael D.

Obesity is a risk factor for cardiovascular disease, hypertension and diabetes. Although a variety vasculogenic factors have been associated with adipose tissue, there is little information available concerning the 3‐dimensional vasculature architecture of adipose tissue. Ten Swiss mice were fed a high fat diet (11% fat content) and ten mice were fed a normal chow diet (4% fat content). After 40 weeks on the diet, the high fat mice (46.7 ± 3.8 gm; mean ± SEM) were significantly larger than the mice on the normal diet (34.5 ± 0.8 gm; mean ± SEM). The mice were anesthetized and the para‐uterine fat pads were infused via the abdominal aorta first with heparinized 0.9% saline, then with 10% buffered formalin and finally with radiopaque silicone rubber (Microfil 122). One para‐uterine fat pad from each mouse was prepared for histological evaluation and the remaining fat pad was prepared for 3‐dimensional micro‐computed tomography. The fat pads were significantly larger in the high fat mice (7.6 ± 1.2 gm; mean ± SEM) than in the normal mice (2.6 ± 0.3 gm; mean ±SEM). There was no difference between the two groups in vascular number density (number of vessels per μl) of the fat pads. However, because of the difference in tissue mass, there was a significant difference between groups in total vessel number. These results indicate that the vascular growth proportionally accompanies tissue growth in adipose tissue. Supported in part by a Faculty Research Grant, Minnesota State University, Mankato, and in part by NIH grant EB 000305.

Despite the importance of the vasculature during tumorigenesis and during antiangiogenesis treatment, there are few methods available to visualize conveniently the three‐dimensional microvascular structure of tumors. Micro‐computed tomography provides a means to visualize conveniently and quantitatively the three‐dimensional tumor microvascular architecture during tumorigenesis and treatment. In the present study, micro‐computed tomography was utilized to visualize the vasculature of hepatic tumors in SV‐40 transgenic rats. The rats were anesthetized and the vasculature of the livers was infused via an aortic cannula, first with heparinized 0.9% saline, then with 10% buffered formalin and finally with radiopaque silicone rubber (Microfil 122). The livers were first scanned by micro‐CT to visualize the microvasculature and then prepared for histology to visualize the parenchyma. At 10 months of age, all transgenic rats developed gross tumors in their livers. Histological examination revealed that the malignant hepatic lesions in the livers displayed mixed structures resembling hepatoblastoma, cholangiocellular or hepatocellular differentiation in human. Various sized vessels were visible extending to the tumors. Three‐dimensional micro‐computed tomography showed that those vessels were disorganized and tortuous with atypical branching patterns. Often vascular “lakes” were found in the mass of the large tumors. These results show the utility of micro‐computed tomography for studying tumor vasculogenesis. Supported in part by NIH grant EB 000305.

Functional renal oxygen consumption, V r O 2 is due to tubular reabsorption of sodium (T Na ) in the proximal (∼67%) and distal(∼30%) nephrons, resulting in compartments (cortex/medulla) with distinct V r O 2 /T Na relationships. A progressive drop in renal blood flow (RBF) with renal arterial stenosis (RAS), disrupts O 2 delivery, vital for reabsorption. The overall objective for the study is to determine the intra‐renal regional alterations in tissue oxygenation (P t O 2 ) and theV r O 2 /T Na relationship in acute RAS. The hypothesis is RAS is accompanied by intra‐renal regional changes in oxygenation. Unilateral acute RAS was induced in 8 pigs by an extra‐vascular balloon. RBF and P t O 2 were measured continuously with an ultrasound flow probe during baseline, RBF autoregulation and 3 sequential decreases in RBF, recovery, and furosemide administration. V r O 2 was determined using Fick arterio‐venous differences. There were concomitant decreases in V r O 2 , and P t O 2 throughout the occlusion periods. Medullary P t O 2 changes were 35% more pronounced than cortical. This study suggests medullary decreases in P t O 2 are more aggravated than cortical changes with reductions in RBF, accentuating the medulla's vulnerability to progressive RAS. Mild to moderately severe obstructions seem to preserve the tubular transport costs. Funding provided by NIH Grant (HL16496‐32), 2007 APS Porter Physiology Fellowship and the Mayo Foundation.

The mechanism of pressure-induced natriuresis remains controversial. To assess whether intracortical or medullary renal blood flows (RBF) change with changes in renal perfusion pressure (RPP), global and regional RBFs were measured using the dynamic spatial reconstructor, a fast computed tomography scanner, in eight anesthetized dogs (group B) within the range of RBF autoregulation (RPP of 153.5 and 114.4 mmHg). Similar measurements were obtained in seven control dogs (group A) in which RPP was not manipulated. In group B, only inner medullary perfusion decreased (from 0.84 to 0.51 ml/min per cm3 of tissue, P = 0.03) with reduction of RPP, whereas global renal, intracortical, and outer medullary perfusions remained unaltered. In group A there was no change in global or regional renal perfusion. The change in inner medullary perfusion in group B (-34.7%) was significantly different (P = 0.021) from that in group A (+27.4%). Global, cortical, and total medullary RBFs (ml/min) and volumes did not change in either group. These results suggest that with changes in RPP, the only detectable change in intrarenal perfusion occurs in the inner medulla.

Appropriate nephron function is dependent on the intrarenal arrangement of blood vessels. The preferred and primary means to study the architecture of intrarenal circulation has been by filling it with opaque substances such as india ink, radio-opaque contrast material, or various polymers for study by light or scanning electron microscopy. With such methodologies, superficial vessels may obscure deep vessels and little quantitative information may be obtained. Serial-section microtomy has not been practical because of problems relating to alignment and registration of adjacent sections, lost sections, and preparation time and effort. Microcomputed tomography (micro-CT) overcomes such limitations and provides a means to study the three-dimensional architecture of filled vessels within an intact rodent kidney and to obtain more quantitative information. As an example of micro-CT's capabilities, we review the use of micro-CT to study the alterations in renal microvasculature caused by the development of liver cirrhosis after chronic bile duct ligation. In this example, micro-CT evidence shows a selective decrease in cortical vascular filling in the kidney, with a maintenance of medullary vascular filling. These changes may contribute to the salt and water retention that accompanies cirrhosis. These results indicate that micro-CT is a promising method to evaluate renal vascular architecture in the intact rodent kidney relative to physiological and pathological function.

Little is known about the distribution of prostaglandin E2 (PGE2) and prostacyclin (PGI2) production in the canine kidney. To determine the basal and stimulated profiles of PGE2 and PGI2 production along the corticomedullary axis of the dog kidney, a slice (0.5 mm thick, 10-50 mg) was obtained from six equally spaced zones along the axis (zone 1, medullary crest; zones 2 and 3, inner medulla; zone 4, outer medulla; and zones 5 and 6, cortex) and was divided into equal halves. One half of the slice was incubated with Krebs-Ringer buffer containing arachidonic acid (6.6 x 10(-4) M), bradykinin (9.4 x 10(-6) M), or indomethacin (10(-5) M), whereas the remaining half of each slice was similarly incubated in Krebs-Ringer buffer alone. The production of PGE2 and 6-keto-PGF1 alpha (the stable metabolite of PGI2) was determined by radioimmunoassay. Under basal conditions, both PGE2 and 6-keto-PGF1 alpha were highest in the innermost zones of the inner medulla (PGE2, 3,328 +/- 549 pg/mg; 6-keto-PGF 1 alpha, 1,611 +/- 129 pg/mg) and decreased exponentially to low levels in the cortex (PGE2, undetectable; 6-keto-PGF1 alpha, 13 +/- 2 pg/mg); this production was inhibited by indomethacin. Arachidonic acid significantly increased the production of PGE2 in all zones of the kidney and the production of 6-keto-PGF1 alpha only in zones 3-6.(ABSTRACT TRUNCATED AT 250 WORDS)

Current microscopic methods to view renal microvasculature reveal only a very limited portion of the total renal volume. Identification of connectivity for postglomerular vessels in the cortex and the medulla during functional states related to changes in sodium excretion will help better to understand the coupling of renal vasculature to tubular function. The purpose of this study was to investigate the possibility of visualizing the continuity of pre- and postglomerular vasculature using three-dimensional micro-computed tomography (micro-CT). Kidneys from normal rats were perfusion fixed in situ at physiological pressure, filled with latex microfil containing lead chromate, and embedded in plastic. The micro-CT scans of the intact kidneys were carried out on a rotating stage illuminated either by a synchrotron x-ray source or a conventional x-ray spectroscopy tube. Images were reconstructed by a filtered backprojection algorithm and volume-rendering techniques were utilized to display the vasculature. The reconstructed images clearly showed the large distribution vessels and the venous drainage of the kidneys, while pre- and postglomerular vessels and their vascular connections throughout the kidney were displayed in great detail. Efferent arterioles showed the characteristics of their peritubular capillary beds in the cortical and medullary regions. The vascular volume of the cortex was 27%, the outer stripe of the outer medulla 18%, the inner stripe of the outer medulla 30%, and the inner medulla 18%. In conclusion, micro-CT is a promising method to evaluate renal vascular architecture relative to physiological and pathological alterations.

Renal function and blood flow decline during aging in association with a decrease in the number of intrarenal vessels, but if loss of estrogen contributes to this microvascular, rarefaction remains unclear. We tested the hypothesis that the decreased renal microvascular density with age is aggravated by loss of estrogen. Six-month-old female C57/BL6 mice underwent ovariectomy (Ovx) or sham operation and then were allowed to age to 18–22 mo. Another comparable group was replenished with estrogen after Ovx (Ovx+E), while a 6-mo-old group served as young controls. Kidneys were then dissected for evaluation of microvascular density (by micro-computed tomography) and angiogenic and fibrogenic factors. Cortical density of small microvessels (20–200 μm) was decreased in all aged groups compared with young controls (30.3 ± 5.8 vessels/mm2, P < 0.05), but tended to be lower in sham compared with Ovx and Ovx+E (9.9 ± 1.7 vs. 17.2 ± 4.2 and 18 ± 3.0 vessels/mm2, P = 0.08 and P = 0.02, respectively). Cortical density of larger microvessels (200–500 μm) decreased only in aged sham ( P = 0.04 vs. young control), and proangiogenic signaling was attenuated. On the other hand, renal fibrogenic mechanisms were aggravated in aged Ovx compared with aged sham, but blunted in Ovx+E, in association with downregulated transforming growth factor-β signaling and decreased oxidative stress in the kidney. Therefore, aging induced in female mice renal cortical microvascular loss, which was likely not mediated by loss of endogenous estrogen. However, estrogen may play a role in protecting the kidney by decreasing oxidative stress and attenuating mechanisms linked to renal interstitial fibrosis.