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INNOVATIVE METHODOLOGY
Departments of Physiology and Biophysics and Medicine, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, California
Submitted 30 December 2005 ; accepted in final form 22 March 2006
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ABSTRACT |
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10-fold the volume of distal tubular fluid, while also causing the release of 20% of juxtaglomerular renin content. Significantly higher speeds of individual red blood cells were measured in intraglomerular capillaries (16.7 ± 0.4 mm/s) compared with peritubular vessels (4.7 ± 0.2 mm/s). Regular periods of glomerular contraction-relaxation were observed, resulting in oscillations of filtration and tubular flow rate. Oscillations in proximal and distal tubular flow showed similar cycle times (45 s) to glomerular filtration, with a delay of 5–10 and 25–30 s, respectively. These innovative technologies provide the most complex, immediate, and dynamic portrayal of renal function, clearly depicting the components and mechanisms involved in normal physiology and pathophysiology. intravital microscopy; diabetic nephropathy; single-nephron glomerular filtration rate; red blood cell velocity; quinacrine; rhodamine; lucifer yellow; proteinuria
Insight into the interplay between different parts of the nephron provides a necessary order of complexity to evaluate and have a comprehensive understanding of renal function. Many critical physiological processes in the kidney like the regulation of glomerular filtration, hemodynamics, concentration, and dilution involve complex interactions between multiple cell types and customarily inaccessible structures. For example, rat experiments in the 1980s revealed that variables of nephron flow exhibited tubuloglomerular feedback (TGF)-mediated regular oscillations (11, 15). Spontaneously hypertensive rats (SHR) have been characterized as displaying irregular TGF-mediated oscillations (11, 31), and recent studies have attempted to distinguish possible factors that may contribute to the spectral complexity of the observed oscillations (14). The existence of TGF-mediated oscillations in nephron flow serves as one example of a complex functional parameter that could be better examined and also quantified by microscopy.
Multiphoton excitation fluorescence microscopy offers a state-of-the-art imaging technique superior for deep optical sectioning of living tissue samples. The higher resolution and minimal phototoxicity of this method permit longer time periods of continuous tissue scanning with uses in real-time imaging of intact organs. Using this technique, dynamic processes such as glomerular filtration (9, 32), proximal tubule endocytosis (23), apoptosis (9), microvascular function (9, 18), protein expression (27), renal cysts (26), and major functions of the juxtaglomerular apparatus, including TGF (20, 22) and renin release (21, 22), have been visualized and studied both in vivo and in vitro down to the subcellular level. 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. In fact, a ratiometric intravital two-photon microscopic technique based on the generalized polarity concept has been recently applied to quantify glomerular filtration and tubular reabsorption (32). The rapidly developing field of fluorescence optics and ultrasensitive detection will fuel further developments. For example, the construction of novel photonic crystal fibers, and hence the advent of multiphoton fluorescence endoscopy (1), demonstrates the potential of this technology for developing noninvasive therapeutic and real-time diagnostic tools for both clinical and biomedical research applications (33). Consequently, one of the next steps for multiphoton microscopy is to provide real-time, quantitative imaging and rapid evaluation of basic organ functions.
The aim of the present study was to extend existing methods of multiphoton fluorescence imaging to directly observe and quantify basic functional parameters of the kidney. These include the noninvasive measurement of glomerular filtration rate, blood flow, urinary concentration/dilution through the course of the nephron, and renin content and release as well as more integrated and complex functions like TGF-mediated oscillations in filtration and tubular flow. These innovative technologies provide the most complex, immediate, and dynamic portrayal of renal function, clearly depicting and analyzing the components and mechanisms involved in normal physiology and pathophysiology.
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MATERIALS AND METHODS |
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Three easy-to-use, water-soluble fluorophores were used to label specific structures in the living kidney. A 70-kDa dextran-rhodamine B conjugate was used (100 µl of a 10 mg/ml stock in iv bolus, Invitrogen) to label the circulating plasma or intravascular space. Tubular segments and, more specifically, the content of individual renin granules were visualized using quinacrine (100 µl of a 25 mg/ml stock in iv bolus, Sigma) in a manner similar to in vitro applications previously described (21, 22). In some experiments, the extracellular fluid marker lucifer yellow (LY) was used (10 µl of a 10 mg/ml stock in iv bolus, Invitrogen). Because the ionic charge of fluorophores affects glomerular filtration characteristics, the FITC-conjugate of the gold-standard glomerular filtration rate (GFR) marker inulin (5 kDa), and the similar neutral rhodamine B-dextran (70 kDa) were used. Although the fluid-marker LY is an anionic compound, it is freely filtered due to its small size (0.45 kDa). All three fluorescent probes were excited using the same, single excitation wavelength of 860 nm (Mai-Tai), and the emitted, nondescanned fluorescent light was detected by two external photomultipliers (green and red channels) with the help of a FITC/TRITC filter block (Leica).
Animals. Munich-Wistar male rats (200 g, Harlan, Madison, WI) and C57/BL6 mice (20 g, inbred) were anesthetized with thiobutabarbital (Inactin, 130 mg/kg body wt) alone (for rats) or in combination with 50 mg/kg body wt ketamine (for mice). After adequate anesthesia was ensured, the trachea was cannulated to facilitate breathing. The left femoral vein and artery were cannulated for dye infusion and blood pressure measurements, respectively. Subsequently, a 10- to 15-mm dorsal incision was made under sterile conditions and the kidney was exteriorized. The animal was placed on the stage of an inverted microscope with the exposed kidney placed in a coverslip-bottomed heated chamber bathed in normal saline, and the kidney was visualized from below as described by Dunn et al. (9) using a HCX PL APO 63X/1.4NA oil CS objective (Leica). High-quality images from the renal cortex were acquired up to 150 µm deep below the surface. During all procedures and imaging, core body temperature was maintained with a homeothermic table. All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Southern California.
In some rats, diabetes was induced by using a single dose of streptozotocin (STZ; 50 mg/kg ip). Blood glucose levels were measured following STZ administration by using test strips on blood samples from a tail clip (Freestyle blood glucose monitoring system, Abbott Laboratories) to verify induction of diabetes. Two groups of animals with blood glucose levels of 400 mg/ml or higher were used: one between days 4 and 6 after STZ injection for SNGFR calculations, and the other within 4 wk for glomerular permeability studies.
During in vivo imaging, the systemic blood pressure of animals was monitored through a cannula inserted into the left femoral artery and the use of an analog single-channel transducer signal conditioner (model BP-1, transducer model BLPR, World Precision Instruments, Sarasota, FL). Calibration was performed using a pressure manometer (model PM-015), and data were collected using data acquisition system QUAD-161.
Chemicals, if not indicated, were purchased from Sigma (St. Louis, MO).
GFR. Overall GFR was measured using the fluorescence-based FITC-inulin method as described before (16). Briefly, anesthetized rats were surgically instrumented for clearance measurements which included tracheostomy, cannulation of left femoral artery and vein as described above, as well as the two ureters using a 24-gauge intravenous (iv) catheter (Terumo). Immediately after surgery, animals were given a bolus (2 µl/g body wt) of PBS containing 0.05% FITC-inulin and 3.5% BSA. This was followed by a maintenance infusion of the same solution at 50 µl/min, and a 30-min equilibration period. Renal function was then determined over three consecutive 15-min clearance periods. At the midpoint of each urine collection, an arterial blood sample (100 µl) was obtained for determination of plasma FITC-inulin. Plasma and urine samples were diluted 1:100 in HEPES buffer, and FITC fluorescence intensity was measured at 540 nm in response to excitation at 485 nm in a cuvette-based fluorometer (Quantamaster-8, PTI). FITC-inulin concentrations were determined using FITC standards (16).
Data analysis. Data are expressed as means ± SE. Statistical significance was tested using ANOVA. Significance was accepted at P < 0.05.
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RESULTS |
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100 µm downstream (ROI2). Internal diameter, length of the tubule, and transit time of filtrate (time shift shown in Fig. 1C) between the two areas were measured using the Quantify package of the Leica confocal software. The midpoint of the dye bolus, approximated by the maximal fluorescence intensity, travels at the same speed as the mean fluid velocity. Thus the transit time (shift between ROI1 and ROI2 intensity plots) was calculated at the peak (Fig. 1C). By calculating tubular fluid volume [length x (diameter/2)2 x 
] the absolute value of SNGFR was calculated (volume/time). Figure 1D demonstrates that SNGFR measurements provided equivalent values if the initial <500-µm segment of proximal tubule was used. There was no statistically significant correlation between SNGFR values and distance of ROIs (r = 0.07). Also, SNGFR values obtained in the same nephrons using LY (32.4 ± 0.4 nl/min, n = 50) or the gold-standard FITC-inulin (34.2 ± 0.8 nl/min, n = 12) were not statistically different.
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Estimating renal blood flow. Red blood cell (RBC) velocity as an index of renal blood flow was measured in peritubular and intraglomerular capillaries by expanding recently established techniques (13, 19, 33). Peritubular capillary blood flow is shown in real time in a supplementary video. Using sufficiently high temporal resolution (1 ms), it was possible to image and characterize the motion of RBCs in the renal cortex. As shown in Fig. 2A, RBCs exclude the fluorescent dye used to label the circulating plasma and consequently they appear as dark, nonfluorescent objects on the images. With the acquisition of repetitive scans along the central axis of a capillary (called a line-scan), the motion of RBCs leaves dark bands in the data set (Fig. 2, A and B). Importantly, the slope of the bands is inversely proportional to the velocity, which was measured as shown in Fig. 2B using the LCS software. In most peritubular capillaries a regular, steady flow of RBCs was measured, as indicated by the constant slope of the bands (Fig. 2, B and C). Significantly higher speeds of RBCs were measured in intraglomerular capillaries (16.7 ± 0.4 mm/s) compared with peritubular vessels (4.7 ± 0.2 mm/s, n = 10 each from 8 different animals) with similar diameters. In addition, we observed instances of irregular flow in some peritubular capillaries in which RBC velocity showed fluctuations (Fig. 2D). Systemic blood pressure was within the normal range throughout these experiments.
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10-fold compared with control. Obstruction of peritubular capillaries by the enlarged distal nephron segments was evident (Fig. 3C). Compared with the magnitude of distal nephron enlargement, no significant morphological changes were observed in proximal tubule segments (Fig. 3). Simultaneously with tubular enlargement, furosemide reduced urinary concentration 50% as measured by the distal/proximal ratio of fluorescence intensity of rhodamine in the tubular fluid (Fig. 3D).
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10 s) and a slower (cycle time 45 s) mechanism. Tubular flow oscillations were delayed compared with oscillations in glomerular filtration: an 5- to 10-s delay was detected in the proximal tubule, whereas the delay was measured to be 25–30 s in distal nephron segments (Fig. 4B). Glomerular and tubular flow oscillations were absent after furosemide treatment (not shown).
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20%, as measured by the reduction in quinacrine fluorescence (Fig. 5C).
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20% of all glomeruli), whereas rhodamine leakage was significantly less noticeable in the hyperfiltering, enlarged glomeruli as illustrated in Fig. 6. The behavior of a 70-kDa dextran-rhodamine conjugate in the vascular space was compared. This large molecule (comparable to the size of albumin) was more freely filtered into Bowman’s space in the sclerotic than in the hyperfiltering glomeruli. The ratio of Bowman’s space/intravascular rhodamine fluorescence was 0.1 ± 0.01 in hyperfiltering glomeruli, whereas it was 0.8 ± 0.1 in sclerotic glomeruli (n = 8 each, P < 0.05). This finding strongly suggests that the significant proteinuria present in diabetic nephropathy is predominantly attributable to the sclerotic rather than the hyperfiltering glomeruli. In most sclerotic glomeruli, the free downstream passage of the "red" proximal tubular fluid was persistent and characteristic. Furthermore, LY given in iv bolus (as shown in Fig. 1) still cleared from Bowman’s space and proximal tubule, evidence that some blood supply and glomerular filtration still remained. |
DISCUSSION |
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In the pioneering work of Molitoris et al. (9, 18, 32), the process of glomerular filtration has been recently observed in real-time using intravital two-photon microscopy and a number of fluorescent probes (9, 32). The presently described methods to quantify the rate of glomerular filtration are based on previous fluorescent techniques that measured SNGFR using conventional micropuncture (16) and tubular flow rate with a nonobstructing optical method (7). In our experience, LY (a widely used extracellular fluid marker) (8) was a better tool to measure SNGFR with this optical technique than FITC-inulin due to its low molecular weight, excellent water solubility, and high fluorescent quantum yield. It should be mentioned, however, that LY is not a good GFR marker in the classic physiological sense (i.e., for clearance studies) because its clearance is most likely greater than that of inulin. Compared with the demanding conventional micropuncture methods (2, 16, 28), a single iv bolus of LY was filtered into the glomeruli and provided a convenient measure of SNGFR within 5 s. Another advantage of this noninvasive technique is that tubular flow and macula densa functions remain intact and undisrupted. Tubular dimensions and the transit time of LY in the early proximal tubule were easily measured using the LCS software (Fig. 1C) from which SNGFR was calculated. Combined with monitoring tubular flow rate (Fig. 4B), this technique permits real-time measurement of SNGFR in the intact kidney. Taking advantage of the high temporal resolution, it was possible to accurately measure LY transit time within the initial 500 µm of proximal tubule. To validate this technique, glomerular filtration was observed and quantified under normal physiological conditions and in diabetic hyperglycemia. Consistent with the well-established glomerular hyperfiltration in diabetes (4, 28), we measured significantly elevated SNGFR levels in select, significantly enlarged glomeruli in STZ-treated animals. The values of SNGFR are within the same range as measured by micropuncture techniques in both control and diabetes (2, 28).
In vivo imaging methods permit the observation and measurement of regional differences in blood flow to the nephron. Cortical blood flow was evaluated by measuring RBC velocity in peritubular and intraglomerular capillaries. Even with the resistance of the afferent arteriole, glomerular capillary pressure is high and the postglomerular resistance produces a significant pressure drop from the glomerular to peritubular capillaries. Thus as expected, RBC velocity was slower in peritubular capillaries than in glomerular capillaries. The occasionally observed irregular blood flow (Fig. 2D) is probably due to intermittent circulation in branches of the peritubular capillary plexus and the dynamic control of vascular resistance upstream. Consistent with the higher blood flow to the kidneys compared with other organs, the measured RBC speed is higher than in other vascular beds (6, 13).
As a noninvasive alternative to existing methods, in vivo imaging allows direct and continuous visualization of all cortical segments of the nephron, often in the same visual field. Such a technique thus permits observation of the ways in which upstream changes exert effects downstream. For example, fluorescence intensity of 70-kDa dextran-rhodamine was more pronounced in distal segments of the nephron, illustrating the concentrating mechanisms present there regionally (Fig. 3). Furosemide, one of the most potent loop diuretics via its ability to block the Na-K-2Cl cotransporter, elicited a massive fluid loss on its acute administration (Fig. 3, B and C). On treatment with furosemide, the increased distal fluid load inflated collecting ducts and diluted the distal tubular fluid (Fig. 3D). The enlarged distal tubular segments appeared to compress peritubular capillaries consistent with preliminary data on the effect of furosemide to reduce renal blood flow (19). Further studies with furosemide would hold the potential to visualize morphological and functional changes occurring in the juxtaglomerular apparatus and glomerulus as well, directly responding to recent inquiries into the functional significance of furosemide’s actions on the Na-K-2Cl cotransporter 1 isoform at these sites (12). Consistent with recent in vivo and in vitro data on the effect of furosemide on renin release (5), the present studies detected a 20% release of renin from juxtaglomerular afferent arterioles in response to acute furosemide treatment (Fig. 5C).
The TGF system is a key regulator of filtration rate and of water and electrolyte delivery to the distal nephron (25). Earlier experiments demonstrated that renal blood flow and consequently, tubular fluid flow, exhibit regular oscillations due to the myogenic mechanism (6- to 10-s periods) and TGF (20- to 50-s periods) (10, 11, 17). Mathematical models as well indicated that these mechanisms are coupled (17). The present experimental technique allows noninvasive, real-time, and in vivo observation and measurement of oscillations in glomerular filtration and tubular flow simultaneously in both proximal and distal nephron segments (Fig. 4B). Future studies can directly visualize alterations in this system, for example, the irregular oscillations in hypertensive rats (11, 14, 31) as well as nephron-nephron interactions (10).
The renin-angiotensin system is one of the most important regulatory mechanisms of renal tubular salt and water conservation, and consequent blood pressure equilibrium. The major structural component of the renin-angiotensin system in the kidney is the juxtaglomerular apparatus located at the vascular pole of the glomerulus. Renin-producing cells of the juxtaglomerular apparatus reside in the wall of the terminal afferent arteriole and can be visualized (Fig. 5, A and B) using multiphoton imaging and quinacrine, a fluorescent probe selective for individual renin granules (21, 22). This model is a direct continuation of our recent in vitro work (21), and it is now possible to study the dynamics of renin content and release in vivo, with high spatial and temporal resolution down to the individual granule level.
The current understanding of diabetes recognizes the disease as a pathology that begins with hyperfiltration that eventually progresses to loss of filtration function. The proteinuria characteristically associated with the disease is considered telltale evidence of concomitant pathology, but the mechanism of its inception was unknown (4). Some theories implicated the hyperfunctioning glomerulus as the culprit: the increased blood flow and vascular damage allowed proteins to leak through (4). Our images and recordings revealed that sclerotizing glomeruli actually leak into Bowman’s space and distal collecting segments, whereas the high-molecular-weight, fluorescence-tagged dextran in the circulating plasma often remains neatly confined within the hyperfiltering glomerular vasculature (Fig. 6). Diabetes has already been implicated in epithelial foot process damage (30), a pathological milestone that would permit proteinuria from the nonfunctional, and hence nondiscriminate, sclerotic glomeruli may be the source of the proteinuria.
In summary, quantitative imaging of the intact kidney with multiphoton microscopy provides an excellent noninvasive tool for visualizing and studying renal function. With the application of only three fluorescent probes, several basic parameters of renal physiology can be measured in real time, including glomerular filtration and permeability, tubular fluid and blood flow, urinary concentration/dilution, and renin content and release. Further studies using this technique will allow investigations of highly complex and integrative questions about the processes of renal (patho)physiology. Quantitative imaging of kidney function with multiphoton microscopy may eventually provide a novel noninvasive diagnostic tool for future clinical applications.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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t) indicates the duration of fluid movement from ROI1 to ROI2. Scale is 50 µm. D: effects of positioning ROI2 at different distances from ROI1 on SNGFR calculations (n = 18).
