Kidney International, Vol. 58 (2000), pp. 1632–1640
Microcomputed tomography of kidneys following chronic bile duct ligation M. CLARA ORTIZ, AGUSTI´N GARCI´A-SANZ, MICHAEL D. BENTLEY, LOURDES A. FORTEPIANI, JOAQUI´N GARCI´A-ESTAN˜, ERIK L. RITMAN, J. CARLOS ROMERO, and LUIS A. JUNCOS Department of Physiology and Biophysics, Mayo School of Medicine, Divisions of Nephrology and Critical Care, Mayo Clinic, Rochester, and Department of Biological Sciences, Minnesota State University, Mankato, Minnesota, USA; and Departamento de Fisiologı´a, Facultad de Medicina, Universidad de Murcia, Murcia, Spain
Microcomputed tomography of kidneys following chronic bile duct ligation. Background. In hepatic cirrhosis, renal sodium and water retention can occur prior to decreases in renal blood flow (RBF). This may be explained in part by redistribution of the intrarenal microcirculation toward the juxtamedullary nephrons. To appreciate this three-dimensional spatial redistribution better, we examined the intrarenal microcirculatory changes using microcomputed tomography (micro-CT) in rats subjected to chronic bile duct ligation (CBDL). Methods. Six kidneys from control rats and eight kidneys from rats that had undergone CBDL for 21 days were perfusion fixed in situ at physiological pressure, perfused with siliconbased Microfil威 containing lead chromate, embedded in plastic, and scanned by micro-CT. The microvasculature in the reconstructed three-dimensional renal images was studied using computerized image-analysis techniques. To determine the physiological condition of the rats, parallel experiments were conducted on six control and six CBDL rats to measure mean arterial pressure (MAP), RBF, glomerular filtration rate (GFR), urine flow (UF) rate, and sodium excretion by conventional methods. Results. The percentage of vasculature in the renal cortex from CBDL rats was significantly decreased (10.8 ⫾ 0.4% vs. 16.8 ⫾ 2.7% control values). However, the vascular volume fractions of the medullary tissues were not significantly altered. There were no significant differences in the number of glomeruli between groups (36,430 ⫾ 1908 CBDLs, 36,609 ⫾ 3167 controls). The CBDL rats had a similar GFR than the controls but a reduced MAP, RBF, UF, and sodium excretion. Conclusions. The results indicate that after CBDL, there is a selective decrease in cortical vascular filling, which may contribute to the salt and water retention that accompanies cirrhosis.
The progression of human and experimental cirrhosis is associated with a spectrum of circulatory and renal Key words: kidney, sodium retention, hepatic cirrhosis, hepatorenal syndrome, renal microcirculation, blood flow redistribution. Received for publication January 13, 2000 and in revised form April 17, 2000 Accepted for publication April 21, 2000
2000 by the International Society of Nephrology
functional alterations [1–5]. There is reduced systemic peripheral vascular resistance and arterial blood pressure and an increased cardiac output [1]; thus, a hyperdynamic circulatory state is present. Despite this hyperdynamic circulation, there is increased renal vascular resistance, renal hypoperfusion [3], and retention of water and salt, which precede the development of ascites [2, 5]. The increase in renal vascular resistance in patients with cirrhosis appears to affect mainly the superficial cortex, sparing medullary blood flow, as suggested by studies using either ultrasound [6, 7] or xenon washout techniques [8]. Thus, because more of the renal blood flow (RBF) is shunted toward the deeper nephrons, which have an enhanced sodium-reabsorbing capability, it is possible that this redistribution is contributing to the enhanced sodium and water retention that is typically seen in cirrhosis [9]. In addition, changes in intrarenal blood flow may be implicated in the progression of human [10] and experimental cirrhosis [11, 12]. Therefore, we think that it is important to evaluate further the abnormalities that are associated with the altered intrarenal distribution of blood flow. In the present study, we determined whether there were abnormalities in the caliber or number of the renal microvessels during chronic bile duct ligation (CBDL). For this, we examined the renal microvasculature of rats that have early renal dysfunction caused by CBDL using microcomputed tomography (micro-CT) technology. The three-dimensional x-ray micro-CT system provides a multidimensional means to obtain qualitative and quantitative information about the vasculature of isolated, fixed rodent organs such as the rat kidney [13, 14]. We then compared these results with a separate group of CBDL rats in which physiologic studies were performed to see whether the changes observed with the microCT were accompanied by changes in RBF, glomerular filtration rate (GFR), and/or sodium and water handling by the kidney.
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METHODS Male Sprague-Dawley rats that were born and raised in the animal facilities of the Universidad de Murcia (Mucria, Spain) were studied by micro-CT. All of the experimental protocols of this study were performed according to the guidelines for the ethical treatment of animals, as specified by the European Union and the Ministerio de Agricultura, Pesca y Alimentacio´n (Spain). All rats were maintained under comparable conditions with an ad libitum diet of normal chow and water. Bile duct ligation Rats initially weighing between 225 and 250 g were subjected to either bile duct ligation or sham surgery as previously described [15]. The rats were anesthetized with an intramuscular injection of a mixture of xylazine (50 mg/kg body wt; Lloyd Laboratories, Shenandoah, IA, USA) and ketamine (100 mg/kg body weight, intramuscularly; Fort Dodge Laboratories, Inc., Fort Dodge, IA, USA). For CBDL surgery, the bile duct was exposed under aseptic conditions by a midline incision under the sternum, doubly ligated with a 2-0 silk suture, and excised between the ligatures. For the sham surgery or control, the bile duct was exposed and dissected, but not ligated or cut. After closure of the abdominal wound, each animal received a subcutaneous injection of Bi-Gentavetina, 0.1 mL/100 g body weight (Schering Plough, Segre, France). All of the experiments were performed after 21 days from the surgery. Physiological study Six CBDL rats and six controls were studied. The rats were anesthetized with thiobutabarbital, 100 mg/kg body weight, intraperitoneally (Inactin, RBI, Natic, MA, USA), and placed on a heated table to maintain body temperature at 37⬚C. A tracheotomy was performed to facilitate ventilation. Polyethylene cannulas were placed in the right femoral artery for measuring blood pressure (HewlettPackard 1280 pressure transducer and amplifier 8805D; Hewlett-Packard, Andover, MA, USA) and withdrawing blood and in the left femoral vein for the infusions. [3H]inulin (1 Ci/mL; New England Nuclear, Itisa, Madrid, Spain) and para-aminohippuric acid (PAH; 0.6 mg/mL; Fluka, Madrid, Spain) were included in the infusion solution (0.9% saline) at a rate of 1.5 mL/h/100 g body weight (Harvard Pump 22; Harvard Apparatus, Boston, MA, USA) for the determination of GFR and RBF, respectively. In addition, the urinary bladder was cannulated to obtain urine samples into preweighed plastic tubes and to calculate the urine flow (UF). Mean arterial pressure (MAP) was recorded continuously throughout the experiment. After a stabilization period of at least 60 minutes, urine was collected for 15 minutes, and blood samples were slowly drawn at the
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midpoint of the 15-minute clearance period. After completion of the experiment, a sample from the renal vein was obtained. Then the anesthetized rat was killed by thoracotomy, and the weight of both kidneys and the spleen was recorded. UF was determined gravimetrically. The concentration of [3H]-inulin in urine and plasma samples was measured using a liquid scintillation counter (Betamatic Basic, Kontron, Madrid, Spain). GFR was calculated as the clearance of radioactive inulin (urine-to-plasma ratio times UF). PAH concentration, in urine (PAHurine) and plasma from the artery (PAHartery) or renal vein (PAHrenal vein), was determined by a colorimetric method [16]. Renal plasma flow (RPF) was calculated as follows: RPF ⫽ PAHurine ⫻ urinary flow/(PAHartery ⫺ PAHrenal vein) Renal blood flow was obtained from the following formula: RBF ⫽ RPF/(1 ⫺ arterial hematocrit) Urinary sodium concentration was measured by flame photometry (Corning 435; Izasa, Barcelona, Spain), and sodium excretion was determined as the product of sodium concentration and UF. All of these variables were factored per gram of kidney weight. Preparation of kidneys for micro-CT scans In this protocol, we used another set of rats to avoid the technical possibility of tissue deterioration that might have occurred over the time course of the physiological study. Eight kidneys were prepared for micro-CT from six CBDL rats and six kidneys from five control rats. The rats were anesthetized with intraperitoneal injection of thiobutabarbital, 100 mg/kg body weight (Inactin, BYK-Gudden, Constance, Germany), and were placed on a heated surgical table to maintain rectal temperature at 37⬚C. To prepare the kidneys for micro-CT, a laparotomy was performed, and a loose ligature was placed around the aorta caudal to the renal arteries. A cannula was connected to a perfusion pump (Syringe Infusion Pump 22; Harvard Apparatus), and a pressure transducer (Recorder 2000; Gould Inc., Instruments Systems Division, Cleveland, OH, USA) was inserted in the aorta at the ligature caudal to the renal arteries. After the cannula was secured in place by the ligature, the MAP was determined. The aorta was then perfused retrograde with a heparinized 0.9% saline solution, and the renal veins were cut to allow venous drainage. The perfusion pressure was maintained at the MAP by adjusting the flow rate of the perfusion pump (approximately 3 mL/ min/100 g). When the perfusate draining through the renal veins was essentially free of blood and the kidneys were uni-
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formly blanched, the kidneys were fixed by continuing the perfusion for five minutes with a 10% neutral-buffered formalin solution (Sigma Diagnostics, St. Louis, MO, USA). The excess of formalin was then removed by continuing the perfusion with 0.9% saline for two to three minutes. Radioopaque Microfil silicone rubber (MV-122; Flow Tech, Carver, MA, USA), containing lead chromate, was then perfused through the aortic cannula. When filling was complete, the kidneys had a uniform deep coloration and the microfil flowed freely from the renal veins. Following perfusion with microfil, the renal arteries and veins were ligated, and the kidneys were removed and weighed. Each kidney was immersed in 70% ethanol and then in aqueous 30, 50, 75, and 100% glycerin solutions, successively in each solution for 24 hours. The kidney was then agitated in 100% acetone for one minute and blotted with a clean dry cloth. Each kidney was embedded in a synthetic resin (Bio-Plastic, Wards Natural Science, Rochester, NY, USA). Micro-CT system The kidneys were scanned using the micro-CT scanner described by Jorgensen, Demirkaya, and Ritman [14]. This scanner consists of a x-ray source, a rotatable specimen stage, a crystaline scintillator, a lens, a charge-coupled device (CCD) camera, and a controlling computer. Scanning was performed by rotating the specimen in specified angular increments about its vertical axis and acquiring a transmitted x-ray image at each increment. The x-ray source in this scanner was a Phillips spectroscopy x-ray tube (PW 2275/20 with a molybdenum anode and a long line focus) with an effective focal spot size of 0.6 ⫻ 0.4 mm. The tube was operated at 35 kev peak and 50 ma providing molybdenum k␣ emission that, when filtered with zirconium foil to remove unwanted higher energy components of the beam’s spectrum, provided a 17.5 kev beam. The specimen stage was positioned 1 m from the x-ray focal spot, minimizing the source’s cone-beam angle and the penumbral blurring. The specimen stage (Newport/Klinger RTN, 120 PP) provided precise rotation to within 0.001 degree and translation to within 0.1 m (Newport/Klinger UT 100, 50 PP). The specimen was rotated at angular increments of 0.499 degrees between views providing 721 views around 360 degrees. The exposure time for each view was 85 seconds. An image of each view was acquired on a clear cesium iodide crystal doped with thallium. A Nikon 50 mm f2.8 enlarger lens was positioned between the scintillator and the CCD camera to provide variable magnification. A CCD camera (TE/CCD-1025 TKB/PI-1, Princeton Instruments, Trenton, NJ, USA) that was cooled to ⫺30⬚C and recorded an image (1024 ⫻ 1024 pixels) at each view. The output images from the CCD were digitized to 16 bits and transferred to the controlling
computer. In this study, all scans were made so that each CCD pixel sampled a square area of 21 m on a side within the specimen. Three-dimensional volume images were reconstructed from the angular views using a modified Feldkamp’s filtered backprojection algorithm [17]. The reconstructed images were generated from cubic voxels, 21 m on a side. The opacity of each voxel was represented by a 16bit gray scale value. Analysis of images Image analysis was carried out using the ANALYZE娃 software package (Biomedical Imaging Resource, Mayo Clinic, Rochester, MN, USA) [18]. This package provides methods to compute, display, and analyze orthogonal and oblique sections from the reconstructed volume images. In addition, the software provides volume-rendering methods to envision the three-dimensional architecture of the renal vasculature. Blood vessel diameters were measured by the Pythagorean theorem in the “calipers” application of the software. Glomerular diameters were determined by counting the number of sections through individual glomeruli and multiplying the number of sections by section thickness. To determine the volumes of the kidneys and their regions, a stereologic application was employed that randomly placed an array of points spaced orthogonally 2.1 mm (that is, 50 voxels, each 42 m) apart throughout the entire scanned volume. The fraction of the volume array that was the tissue of interest was determined by counting the number of points within the boundary of the tissue and dividing by the total number of points. The tissue volume was then determined by multiplying the volume fraction of the tissue of interest by the total scanned volume of the image [19]. The vascular volume fraction in each part of the renal tissue was determined following the method of Hillman et al [20]. The determinations were made from midtransverse slices that included the cortex, the outer stripe of the outer medulla (OSOM), the inner stripe of the outer medulla (ISOM), and the inner medulla (IM). The average opacity was measured from regions of interest within a large interlobar artery (Oartery), in the tissues (Otissue), and in the background matrix outside the kidney (Obg). The vascular volume fraction in each tissue was determined as follows: (Otissue ⫺Obg)/(Oartery ⫺ Obg) Glomeruli were counted using the disector principle of Sterio [21]. For our study, the disector was comprised of a counting field that had an area of 1.6 mm2 and a corresponding reference field on a parallel section that was spaced 0.063 mm from the counting field. A glomerulus was counted in the counting field if it did not touch the lower or left margins of the counting field and if it did not appear in the reference field. The glomeruli were
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Ortiz et al: Renal micro-CT following bile duct ligation Table 1. Renal function of rats that had undergone chronic bile duct ligation (CBDL, N ⫽ 6) or sham surgery (controls; N ⫽ 6) Body weight
Kidney weight g
Control CBDL
299.5 ⫾ 2.9 276.8 ⫾ 11.1
RBF MAP mm/Hg
2.31 ⫾ 0.13 2.19 ⫾ 0.04
120.9 ⫾ 5.3 102.6 ⫾ 4.8a
GFR mL/min/g
6.30 ⫾ 0.31 5.02 ⫾ 0.59a
0.96 ⫾ 0.14 0.80 ⫾ 0.18
UF lL/min/g
Sodium excretion lEq/min/g
57.37 ⫾ 2.46 15.29 ⫾ 3.30a
3.29 ⫾ 0.85 1.78 ⫾ 0.36a
Data are expressed as mean ⫾ standard error of the mean a CBDL vs. control, P ⬍ 0.05
counted in four disectors in each of 13 cortical samples, each sample having a volume of 0.4 L (4 ⫻ 1.6 mm2 ⫻ 0.063 mm). The 13 cortical samples were taken systematically from each kidney. One sample was taken from each pole. Three samples were taken from the numeric midregion of the kidney, and four samples were taken from each of the two numeric quartile regions located midway between the poles and the midregion. Thus, for each kidney, 200 to 300 glomeruli were counted in a total sample volume of 5.2 L (13 ⫻ 0.4 L). Statistical analysis
Table 2. Tissue composition of kidneys from rats that had undergone chronic bile duct ligation (CBDL; N ⫽ 8) or sham surgery (controls, N ⫽ 6) Tissue Cortex OSOM ISOM IM Whole kidney
Experimental condition Control CBDL Control CBDL Control CBDL Control CBDL Control CBDL
Tissue volume lL 776.0 ⫾ 54.1 752.1 ⫾ 33.5 315.4 ⫾ 11.9 310.6 ⫾ 16.8 171.6 ⫾ 12.0 145.7 ⫾ 15.5 75.3 ⫾ 12.5 65.4 ⫾ 4.4 1392.5 ⫾ 83.4 1319.3 ⫾ 65.0
Tissue volume fraction % 55.6 ⫾ 0.6 57.2 ⫾ 1.3 22.8 ⫾ 0.8 23.7 ⫾ 1.0 12.3 ⫾ 0.5 10.9 ⫾ 0.6 5.3 ⫾ 0.6 4.9 ⫾ 0.2
Data are reported as mean ⫾ SEM. Student’s unpaired t-test was used to compare values obtained from kidneys of bile duct-ligated rats to values obtained from control rats. Differences were considered significant if P ⬍ 0.05.
Data are expressed as mean ⫾ standard error of the mean. Abbreviations are: OSOM, outer stripe of the outer medulla; ISOM, inner stripe of the outer medulla; IM, inner medulla. There were no significant changes.
RESULTS
kidneys was similar to controls (Table 2). Accordingly, the volumes of the cortical and medullary tissues in the CBDL kidneys were not significantly different from the corresponding tissues in the controls (Table 2). Because of the similarity in tissue volumes between both groups, the corresponding tissue volume fractions were also similar (Table 2). The images obtained by micro-CT are three-dimensional arrays of cubic voxels having opacities that represent the amount of microfil in the vascular compartment. In both groups of kidneys, all regions were opacified, and there were no regions that would indicate that the main vascular distribution branches to the tissues were obstructed during preparation. Likewise, in every kidney, the arteries and their corresponding veins were opacified with microfil, indicating that the entire renal circulation had been filled. As a further indication of complete filling, the interlobar veins were distended, and the average diameter was significantly larger than that the interlobar arteries. Although the diameter of the interlobar arteries in the CBDL kidneys (371 ⫾ 8 m) was not significantly different from those of the controls (353 ⫾ 15 m), the diameter of the interlobar veins was significantly larger in the CBDL kidneys (897 ⫾ 17 m) than in the controls (793 ⫾ 21 m). In sections taken from the image arrays, the different kidney regions in both experimental groups were easily
Chronic bile duct ligation rats had abdominal distention and jaundice with yellow ears, tails, and paws in comparison to the normal pink coloration found in the control rats. The urine of the CBDL rats was intense yellow in comparison to the weak-yellow urine of the controls. Spleens and livers of CBDL rats were enlarged, and the livers were hard, with a pulpy texture and a granular tan-brown coloration. After the blood was flushed from the vasculature, the color of the CBDL kidneys was green, whereas the color of controls was white. Physiological study Renal function was measured in six CBDL and six control rats (Table 1). MAP and RBF in the CBDL rats were significantly less than the values measured in the control rats. Although there was no significant difference in GFR between both groups, the UF and sodium excretion were significantly reduced in the CBDL rats. Micro-CT study The kidney weight of the CBDL rats (1.96 ⫾ 0.04 g) was not significantly different from the controls (1.86 ⫾ 0.12 g). Volumetric measurements of images obtained by micro-CT indicated that the volume of the CBDL
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Fig. 1. Midcoronal 42 m thick slices selected from a reconstructed image of a kidney from a chronic bile duct-ligated (CBDL) rat (left) and a control (right). The contrast range of both images is the same. Abbreviations are: Cort, cortex; OSOM and ISOM, outer and inner stripe of the outer medulla, respectively; IM, inner medulla.
identified by their characteristic vascular features. The cortex was characterized by the presence of glomeruli and the medulla by parallel bundles of vasa recta. OSOM, ISOM, and IM were identified by the density of the vasa recta bundles (Fig. 1). The images of the CBDL kidneys appeared to be strikingly different from images of the control kidneys. Specifically, the cortex of the CBDL kidneys was less opaque than the control tissue, whereas the outer and inner medulla of the CBDL kidneys had similar opacities to those of the controls. Although glomeruli were visible in the cortex of both groups of kidneys, the glomeruli appeared to be less opaque in the CBDL group than in the control. Because the microfil remains within the vascular compartment, the opacity of the tissue represents the amount of opacified vasculature in the tissue. While there was no significant difference between the CBDL and control kidneys in the vascular volume fractions of the medullary tissues, the vascular volume fraction of the cortex in the CBDL kidneys was significantly different from that of the controls (Fig. 2). The percentage of filled vasculature (vascular volume fraction) in the cortex was 10.8 ⫾ 0.4% in CBDL kidneys and 16.8 ⫾ 2.7% in controls. The vascular volume fraction of both the glomerular and the peritubular capillary beds of the CBDL kidneys were significantly different from those of the control kidneys (Table 3). No significant differences were observed between CBDL and control kidneys in glomerular diameter or in number of glomeruli (Table 3). DISCUSSION The purpose of this study was to examine the renal microvasculature from rats subjected to CBDL by using
Fig. 2. Vascular volume fraction of different regions of the kidneys from rats subjected to CBDL (䊏; N ⫽ 8) or controls ( ; N ⫽ 6). *Significantly different from control, P ⬍ 0.05.
a three-dimensional micro-CT system. The main finding of our study was decreased filling of the cortical microvasculature in the kidneys, suggesting that there is hypoperfusion in the renal cortex 21 days after bile duct ligation. The medullary vasculature, on the other hand, did not appear to be affected. These changes in the renal microvasculature may contribute to the sodium and water retention found at this stage of CBDL. Biliary obstruction of more than 14 days in rats leads to morphological abnormalities in the liver that are associated with portal hypertension [22, 23]. However, the time of development of secondary biliary cirrhosis appears to vary widely. These variations may be attributed to a number of factors such as the rat strain [24–26], the use of anesthesia [27], and the duration of bile duct obstruction [28, 29]. In the present study, the rats with
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Table 3. Glomerular data of kidneys from rats subjected to chronic bile duct ligation (CBDL; N ⫽ 8) or sham surgery (controls, N ⫽ 6)
Control CBDL
Intraglomerular vascular volume
Diameter lm
Number per l cortex
Number per Kidney
130 ⫾ 5 133 ⫾ 5
47.5 ⫾ 3.1 48.9 ⫾ 2.8
36,609 ⫾ 3,167 36,430 ⫾ 1,908
Interglomerular vascular volume %
27.6 ⫾ 2.0 20.4 ⫾ 0.9a
13.6 ⫾ 1.5 8.0 ⫾ 0.5a
Data are expressed as mean ⫾ standard error of the mean. a Significantly different from control, P ⬍ 0.05
CBDL did not develop ascites; however, they exhibited enlargement and altered appearance of the liver, splenomegalia, and splanchnic vasodilation that confirm the presence of chronic liver disease. Furthermore, in comparison to control rats, CBDL rats exhibited reduced MAP, RBF, and urinary sodium and water excretion, although GFR was relatively preserved. These results are in agreement with other studies in which rats do not typically develop ascites until four weeks after the bile duct ligation [25, 29, 30]. The CBDL animals had a marked reduction in the vascular volume of the cortex that was accompanied by a relatively small reduction in total RBF; the medullary vascular volume was maintained. This alteration in the intrarenal vascular filling could not be accounted for by the flushing and fixing of the kidney because the control animals were subjected to the same procedure and did not exhibit the reduction in cortical vascular filling. The reduction in the cortical vascular volume fraction from CBDL kidneys suggests that there was either a reduction in the number of vessels in the cortex or a decrease in the diameter of the vessels. Because we found similar number of glomeruli in both groups of kidneys, but a lower cortical vascular volume fraction in both intraglomeruli and interglomeruli areas in the CBDL kidneys, it is likely that the predominant mechanism for the reduced vascular volume was a decrease in the diameter of the vessels in the renal cortex, rather than a reduction in the number of vessels. The probable reduction of the vascular diameters in turn could be due to either decreased distending pressure within the vessels (that is, low blood pressure) or increased cortical vascular resistance (caused by either structural changes or vasoconstriction of the cortical vessels). We do not think that a decrease in the distending pressure (despite the lower blood pressure of the CBDL rats) accounts for the decreased vascular filling for several reasons. First, the most likely response to decreased distending pressure would be myogenic-induced vasodilation, that is, an increase rather than a decrease in microvascular diameters (unless autoregulation is impaired). Second and more important, if the distending pressures were so low so that the diameter of the microvessels decreased, one would expect that the diameters of the larger arteries would also decrease.
However, the vascular diameters of the interlobar arteries were not different than those in the control group, suggesting that the distending pressures were not causing the difference in vascular filling, and also that the renal vasoconstriction was limited to the cortical resistance vessels. Finally, one would have expected a drastic decrease in RBF if the distending pressures were that low, whereas we found only a modest decrease. As mentioned previously in this article, the changes in vascular diameter could be due to an increase in vascular resistance. It could be argued that because we are flushing and fixing the kidney, we would not be able to detect changes in vascular resistance with our preparation. However, Cuttino et al used a similar preparation (ex vivo microradiography with a nearly identical flushing/ fixing technique) and found changes in vascular filling in the vasa recta and postglomerular capillary bed two hours after infusion of bromoethylamine hydrobromide [31]. This indicates that changes in vascular resistance may remain present when the tissue is rapidly fixed prior to infusion of the contrast media. Moreover, the changes in vascular resistance that we observed are likely due to prolonged vasoconstriction (that may not be readily reversed by a vasodilator stimulus) or a structural change (hyperplasia/hypertrophy of the vascular media causing a decreased lumen and thus increased resistance) such as that seen in hypertension. Indeed, this may explain why there was a prolonged delay before renal function recovers when kidneys from patients with hepatorenal syndrome are transplanted into patients without liver disease [32]. Despite the marked decrease in cortical vascular filling, the decrease in RBF was smaller than expected. In this respect, it is important to note that the vascular volume is not a direct measure of blood flow. This is because blood flow velocity may be increased through the smaller diameters, which would tend to sustain blood flow (as occurs during autoregulation). However, in our experiments, blood flow velocity was unlikely increased because blood pressure was decreased. Thus, it is probable that the decreased cortical vascular volume represented a fall in cortical blood flow. Indeed, previous studies have also suggested decreased cortical blood flow [33]. Moreover, despite the fact that vascular volume is
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not a measure of blood flow, it does tend to be a marker of blood flow under many physiologic circumstances. Fast computerized tomography studies in the dog have shown that as RBF is reduced, there is a corresponding decrease in cortical blood flow, which is more dependent on reduced vascular volume than transit time [34]. A second possible explanation for the apparent discrepancy between the preserved RBF in the presence of decreased cortical vascular volume is suggested by the preserved medullary vascular filling. This preservation of medullary vascular filling in the presence of decreased cortical vascular filling suggests that the medullary blood flow may be enhanced (thus tending to preserve total RBF). This redistribution of intrarenal blood flow has been described in a variety of conditions such as reduction of renal artery perfusion pressure [35], hemorrhagic hypotension [36], ureteral occlusion [37], sodium restriction [38], and renal venous pressure elevation [39]. Cortical renal vasoconstriction has also been documented in human cirrhosis using different techniques [6, 8, 9]. Gines, Martin, and Niederberger have proposed that the existence of renal vasoconstriction, in the absence of morphological changes in the kidney, is highly suggestive of alterations in vasoactive factors acting on renal circulation [40]. The activation of vasoconstrictor and antinatriuretic systems (for example, renin-angiotensin, sympathetic nervous system, arginine vasopressin) as a consequence of the disturbance in the systemic circulation could play a critical role in the pathogenesis of this disorder. For instance, the medullary circulation might be preserved due to specific activation of medullary vasodilators such as prostaglandin E2 and/or nitric oxide [3, 6, 41]. Conversely, cortical vasoconstriction may be due to agents that either act on or are located predominantly in the cortex [3, 41]. For example, the reninangiotensin system is activated in human and experimental cirrhosis [42]. Indeed, Ubeda et al have shown an increased renin-mRNA levels in kidneys from rats three weeks after bile duct ligation, indicating hyperactivity of the juxtaglomerular cells [15]. Because there is a transcortical gradient of renin, with greater quantities of renin present in superficial than in juxtamedullary glomeruli [43], it is possible that the increased levels of renin (and angiotensin II) present in the cortex cause a predominant cortical (local) vasoconstriction in CBDL, thus contributing to the vascular redistribution. In this respect, it is interesting to note that RBF fell to a greater degree than GFR, suggesting predominant constriction of the efferent arterioles. Because angiotensin II may preferentially constrict the efferent arteriole [44], the relative preservation of GFR may also support an increase in renal angiotensin II activity. Furthermore, Bank and Aynedjian [24] and Monasterolo, Peiretti, and Elias [28] provide evidence that an efferent vasoconstriction occurs in bile duct-ligated rats, which may account for an altered
sodium excretion by the kidney. Because these studies were performed 6 and 14 days after bile duct ligation, respectively, the results suggest that the cortical vascular alterations may occur in early phases of the disease. However, one must take into account that changes in RBF and GFR do not necessarily correlate very well with the degree of vasoconstriction [40], and changes in Kf can also have an important impact on GFR; thus, one must interpret these findings with caution. An alternative possibility for the renal abnormalities relates to the renal venous pressures. We observed an increased diameter in the interlobar veins from CBDL kidneys, likely a consequence of elevated resistance to portal flow in the CBDL animals: Increased total venous capacity secondary to increased portal pressure has been reported in cirrhotic rats without ascites [45]. Furthermore, increased renal venous pressure has been reported in human cirrhosis [46]. The increase in renal venous pressure can affect the microvascular hemodynamic and tubular function in the kidney [47]. In fact, as mentioned previously in this article, renal venous pressure elevation can cause redistribution of RBF and sodium retention. It is interesting to note that this condition (as well as most others associated with intrarenal redistribution of blood flow) is accompanied by increased renin, further suggesting a role for the renin-angiotensin system. However, this possibility clearly requires further study. It should be noted that previous reports examining papillary blood flow showed a significant decrease in papillary blood flow in cirrhotic rats [25, 48]. The reasons for this discrepancy are unclear, but may be explained by the following reasons: First, the model of cirrhosis was different. Cirrhosis was induced by carbon tetrachloride rather than bile duct ligation. Second, the strains of rats were different: Munich-Wistar in the previous studies versus Sprague-Dawley in the present one. Third, at least in one of the studies [25], the animals had a more advanced stage of cirrhosis with ascites, which may be associated with a more diffuse decrease in perfusion to all segments of the kidney. Finally, the methods for measuring papillary blood flow were different. The previous studies used laser-Doppler flowmetry, which records changes in RBC velocity from a localized area of the papilla, whereas micro-CT provides a more global assessment of papillary circulation. The distribution of intrarenal blood flow has been implicated in physiologic regulation of renal function. Not only does nephron blood flow alter GFR, but it can also regulate peritubular capillary uptake by changing the colloid osmotic and hydrostatic pressures. In addition, changes in regional blood flow, by shunting blood flow toward the juxtamedullary nephrons (as seen in our present study), could be a factor in the renal modulation of sodium excretion. This may be because long-looped nephrons of the deep cortex may have a slow fluid transit
Ortiz et al: Renal micro-CT following bile duct ligation
time and thereby a higher capability to reabsorb sodium. The micro-CT can only determine the vascular volume but not the transit time. Thus, further studies are needed to address this possibility. Regardless, our results suggest that the new tubular–vascular relationship could contribute to sodium retention in the rats subjected to CBDL. The implications of this study are that in the progression of cirrhosis after bile duct ligation, there is a decrease in RBF that shifts the distribution of the renal microcirculation from the cortex to the medulla, favoring sodium and water retention from long-looped nephrons. Systemic and intrarenal vasoactive factors could influence in the intrarenal redistribution of blood flow and would further potentiate the reabsorption of sodium and water in the cirrhosis. ACKNOWLEDGMENTS This work was supported, in part, by NSF grant BIR9317816, NIH grants RR11800 and HL16496, and SAF97-0176 from the Spanish Health and Pharmacy National Plan (J.G.E.). Dr. Ortiz was supported by a fellowship of the Spanish Society of Hypertension donated by Merck Sharp & Dohme. Dr. Garcı´a-Sanz was supported by the National Committee for the Development of Science and Technology of Venezuela (CONICIT) and Elmor Laboratories. The authors are grateful to Mr. Steven Jorgensen, Ms. Patricia Lund, and Ms. Denize Reyes for their technical assistance; to Ms. Kristy Zodrow for preparation of this manuscript; and to Ms. Julie Patterson for preparation of the illustrations. Reprint requests to J. Carlos Romero, M.D., Department of Physiology and Biophysics, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, USA. E-mail:
[email protected]
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