Am J Physiol Renal Physiol 291: F495–F502, 2006.
First published April 11, 2006; doi:10.1152/ajprenal.00521.2005.
Innovative Methodology
Quantitative imaging of basic functions in renal (patho)physiology
Jung Julie Kang, Ildiko Toma, Arnold Sipos, Fiona McCulloch, and Janos Peti-Peterdi
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
RECENT ADVANCES HAVE ADDED microscopic investigative techniques to the arsenal of tools applicable to gaining a more
comprehensive understanding and quantifiable benchtop assessment of renal function. The development of micropuncture
by Wearn and Richards (29) in 1924 was one of the most
significant advances in renal physiology, leading to the discovery of the phenomena of filtration, absorption, and secretion in
the nephron (24). The localization of these processes and the
determination of the handling of specific substances were
deduced by comparing fluid compositions in the bladder,
kidney, and blood (29). Eventually, manipulation of renal
physiological functions was made possible with the development of the isolated perfused tubule by Burg and colleagues (3)
in 1966. Micropuncture and microperfusion techniques can be
used to study the end results of renal physiological function,
but they do not permit direct visualization of ongoing mechanisms or other details of the pathways. Renewed interest in
employing conventional methods to study nephron function in
genetically altered animal models has prompted innovations
like the use of FITC-labeled inulin, rather than radioactive
probes, to evaluate single-nephron glomerular filtration rate
(SNGFR) and tubular reabsorption (16). The application of
fluorescence microscopy to determine the FITC-inulin signal in
micropuncture studies has the advantages of simplicity, precision, accuracy, and cost effectiveness without consumption of
the fluid sample (16). These long-established, demanding, and
invasive methods provide critical snapshots of renal physiology and have directed countless current methods to discover
precise details about the players, interactions, and control
mechanisms involved.
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
Address for reprint requests and other correspondence: J. Peti-Peterdi, Univ.
of Southern California, Zilkha Neurogenetic Institute, 1501 San Pablo St., ZNI
335, Los Angeles, CA 90033 (e-mail: [email protected]).
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.
intravital microscopy; diabetic nephropathy; single-nephron glomerular filtration rate; red blood cell velocity; quinacrine; rhodamine;
lucifer yellow; proteinuria
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0363-6127/06 $8.00 Copyright © 2006 the American Physiological Society
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Kang, Jung Julie, Ildiko Toma, Arnold Sipos, Fiona McCulloch,
and Janos Peti-Peterdi. Quantitative imaging of basic functions in renal
(patho)physiology. Am J Physiol Renal Physiol 291: F495–F502, 2006.
First published April 11, 2006; doi:10.1152/ajprenal.00521.2005.—Multiphoton fluorescence microscopy offers the advantages of deep optical sectioning of living tissue with minimal phototoxicity and high
optical resolution. More importantly, dynamic processes and multiple
functions of an intact organ can be visualized in real time using
noninvasive methods, and quantified. These studies aimed to extend
existing methods of multiphoton fluorescence imaging to directly
observe and quantify basic physiological parameters of the kidney
including glomerular filtration rate (GFR) and permeability, blood
flow, urinary concentration/dilution, renin content and release, as well
as more integrated and complex functions like the tubuloglomerular
feedback (TGF)-mediated oscillations in glomerular filtration and
tubular flow. Streptozotocin-induced diabetes significantly increased
single-nephron GFR (SNGFR) from 32.4 ⫾ 0.4 to 59.5 ⫾ 2.5 nl/min
and glomerular permeability to a 70-kDa fluorophore approximately
eightfold. The loop diuretic furosemide 2-fold diluted and increased
⬃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.
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MATERIALS AND METHODS
Multiphoton excitation laser-scanning fluorescence microscopy.
The multiphoton microscope used in these studies consists of a Leica
TCS SP2 AOBS MP confocal microscope system (Leica Microsystems, Heidelberg, Germany). A Leica DM IRE2 inverted microscope
was powered by a wide-band, fully automated, infrared (710 –920 nm)
combined photo-diode pump laser and mode-locked titanium:sapphire
laser (Mai-Tai, Spectra-Physics, Mountain View, CA). Images were
collected in a time (xyt, 20 Hz) or line (xt, 1,000 Hz) series, depending
on the purpose of study, with Leica confocal software (LCS
2.61.1537) and analyzed with the LCS 3D, Process, and Quantify
packages. The principles and advantages of conventional confocal and
two-photon microscopy and their application in imaging renal tissues
have recently been reviewed (18, 22).
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 ␮l 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 ␮l 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 ␮l 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
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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 coverslipbottomed 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 ␮m 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 ␮l/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 ␮l/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 ␮l) 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.
RESULTS
Measurement of SNGFR. An example of the experimental
technique is shown in Fig. 1. A superficial glomerulus was
selected that had at least a 100-␮m-long initial segment of the
proximal tubule positioned in the same optical section. A
single iv bolus of the fluid marker LY was injected into the
femoral vein, which appeared in the glomerulus within 5 s and
filtered into Bowman’s space and early proximal tubule (see
supplementary video file showing glomerular filtration of LY,
as well as all supplemental data for this article in the online
version of this article). Using high temporal resolution, the
fluorescence intensity changes of LY were measured as shown
in Fig. 1, A–C, within two regions of interest (ROI): one at the
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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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opening of proximal tubule (ROI1), the other ⬃100 ␮m 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 ⫻ (diameter/2)2 ⫻ ␲] the absolute value of SNGFR
was calculated (volume/time). Figure 1D demonstrates that
SNGFR measurements provided equivalent values if the initial
⬍500-␮m 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.
To demonstrate the utility of this technique, SNGFR was
measured in control and STZ-diabetic rats. SNGFR in control
rats averaged 32.4 ⫾ 0.4 nl/min (n ⫽ 50 from 7 different
animals), whereas significantly elevated levels (59.5 ⫾ 2.5
nl/min, n ⫽ 15 from 7 different animals) were measured in
select, significantly enlarged glomeruli from diabetic rats.
Overall GFR measured with the FITC-inulin clearance method
appeared to be higher in diabetes, but there was no statistically
significant difference between the control (1.13 ⫾ 0.13 ml/min,
n ⫽ 6) and diabetic groups (1.34 ⫾ 0.13 ml/min, n ⫽ 5).
Systemic blood pressure was normal in all animals studied; the
mean arterial blood pressure was 102.7 ⫾ 8.6 mmHg.
Estimating renal blood flow. Red blood cell (RBC) velocity
as an index of renal blood flow was measured in peritubular
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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.
Imaging urinary concentration and dilution. We applied
multiphoton microscopy to provide a continuous, real-time
measurement of the urinary concentrating and diluting function
of individual renal tubules based on recent work (26). The
technique is demonstrated in Fig. 3. A single iv bolus of a
relatively high-molecular-weight fluorescent plasma marker
(rhodamine-conjugated 70-kDa dextran) remained in the intravascular space for several hours of imaging due to its large
size. Steady-state levels of the marker in the plasma were
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Fig. 1. Representative images of the technique to measure
single-nephron glomerular filtration rate (SNGFR). The
intravascular space is labeled with 70-kDa dextran-rhodamine B (red), renal tubules with quinacrine (green), and
the glomerular filtrate (tubular fluid) with lucifer yellow
(LY; yellow). Glomerulus (G) and proximal tubule (PT)
are labeled. Fluorescence intensity of intravenously injected LY was measured within 2 areas of interest (ROI) in
the early PT fluid: one always at the beginning of the PT
(ROI1), the other ⬃100 ␮m downstream (ROI2). A and B:
downstream movement of filtered LY in tubular fluid. C:
plot of LY fluorescence intensity changes within the 2
ROIs. The shift (⌬t) indicates the duration of fluid movement from ROI1 to ROI2. Scale is 50 ␮m. D: effects of
positioning ROI2 at different distances from ROI1 on
SNGFR calculations (n ⫽ 18).
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evident by the constant level of fluorescence in the renal
vasculature throughout these experiments (Figs. 1– 6, supplementary videos). A microscopic fraction of the marker was
filtered in the glomeruli, and thus only trace amounts of
rhodamine were detected in the proximal tubular fluid (see dark
proximal tubule fluid in Fig. 3A). However, due to the concen-
trating mechanism, an approximately sevenfold higher level of
rhodamine fluorescence was present in the collecting duct
system (Fig. 3, A and D). Subsequently, the loop diuretic
furosemide was used to demonstrate the capabilities of this
quantitative imaging technique. Effects of a single iv bolus of
2 mg/kg furosemide on the morphology and function of the
Fig. 3. Visualization of the concentrating and diluting
function of the kidney. A: circulating plasma was labeled
with 70-kDa dextran-rhodamine B (red) and PT and DT
(*) with quinacrine (green). Note that some rhodaminedextran was filtered through the glomerulus (G) and became concentrated in the DT vs. PT as indicated by the
intense red color of tubular fluid. B and C: effects of the
loop diuretic furosemide. Note the reduced red color and
increased volume of DT fluid. D: reduction in the ratio of
DT/PT fluid rhodamine B fluorescence in response to
furosemide, injected intravenously at time 0. Scale is 20
␮m.
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Fig. 2. Illustration of red blood cell (RBC) flow and velocity measurement in renal capillaries. A: circulating plasma was labeled with 70-kDa dextran-rhodamine
B (red) and proximal (PT) and distal (DT) renal tubules with quinacrine (green). Note that some rhodamine was filtered through the glomerulus and became
concentrated in the DT as indicated by the red color of tubular fluid. A horizontal line, ⬃20 ␮m long, was positioned through the central axis of a capillary (white
line in the top left corner), and a line (xt) scan was performed at 1-ms intervals along this line continuously for 2 s. RBCs appear as unstained dark objects (A)
and leave dark bands on the line scan (B–D) as they move through the capillary (line). Xt line scans of a peritubular (B) and intraglomerular (C) capillary are
shown. RBC velocity as the slope of the bands ⌬x/⌬t was calculated as shown. Note the regular blood flow (constant slope) in B and C and higher RBC speed
(smaller slope) in C vs. B. D: line scan of irregular blood flow in a peritubular capillary, indicated by the variable slope.
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renal cortex are shown in Fig. 3, B–D. Within 2 min of
administration, furosemide caused the enlargement of distal
nephron segments (Fig. 3B). At the peak of furosemide’s
effect, the volume of tubular fluid in the cortical collecting
tubule increased ⬃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).
Physiological oscillations in SNGFR and tubular flow. In the
intact kidney, the closed loop system allows for the observation
of myogenic and TGF-induced oscillations in GFR and, consequently, changes in tubular flow. The renal cortex was
visualized continuously for 5 min, as illustrated in Fig. 4. A
video of the same preparation is available at http://ajprenal.
physiology.org/cgi/content/full/DC1. Regular periods of glomerular contraction-relaxation were observed, resulting in oscillations of filtration and tubular flow rate (Fig. 4B). This was
measured by the changes in fluorescence intensity of the
filtered plasma marker, 70-kDa dextran-rhodamine B, in the
tubular (Bowman’s space) fluid as well as by changes in
tubular diameter (not shown; see supplementary video). Oscillations clearly showed two components: a faster (cycle time
⬃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).
Renin content and release. Renin content of juxtaglomerular
granular cells was visualized in vivo in the rat (Fig. 5A) and
mouse (Fig. 5B) using quinacrine, a selective marker of acidic
and secretory granules. Quinacrine staining of individual renin
granules in the terminal afferent arteriole was highly specific.
However, weak background staining was observed in all tubule
cell types, most likely due to the accumulation of quinacrine in
lysosomes. In addition to the quantification of renin content
(based on the length of the renin-positive segment of afferent
arteriole), we measured the dynamics of renin release. Furosemide was used to trigger juxtaglomerular renin release.
Figure 5C shows a representative graph of four separate ex-
Fig. 5. In vivo imaging of the juxtaglomerular renin content in the rat (A) and mouse (B). Circulating plasma is labeled with rhodamine B-conjugated 70-kDa
dextran (red), content of individual renin granules with quinacrine (green), and nuclei with Hoechst 33342 (blue). Note the significant renin content around the
juxtaglomerular portion of the afferent (AA), but not the efferent arteriole (EA). G, glomerulus. Scale is 20 ␮m. C: reduction in quinacrine fluorescence intensity
(index of renin release) in response to furosemide, injected intravenously at time 0.
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Fig. 4. Fluorescence labeling of the intact living rat kidney. A: rhodamine
B-conjugated 70-kDa dextran (red) was given intravenously to label the
cortical vasculature (plasma). Quinacrine (green) stained renal tubules [PT and
cortical collecting duct (CCD)]. A small fraction of plasma dextran is filtered
in the glomerulus (G) and appears in the concentrated tubular fluid of the CCD
(red) more or less intensely depending on the high or low phase of flow
oscillations (see supplementary video). B: spontaneous oscillations in glomerular filtration and PT and DT flow detected by changes in rhodamine B
fluorescence in the tubular fluid (or Bowman’s space). Magnitude of changes
are not drawn to scale. Simultaneous measurements are shown (vertical dotted
line represents time 0), so the delay in PT and CCD oscillations compared with
those in the parent glomerulus (Glom) is easy to read. Scale is 20 ␮m.
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periments. Minutes after iv bolus administration of 2 mg/kg
furosemide, the renin content of the afferent arteriole was
reduced by ⬃20%, as measured by the reduction in quinacrine
fluorescence (Fig. 5C).
Glomerular permeability. In vivo real-time imaging allowed
us to observe functional differences between hyperfiltering and
sclerotic glomeruli in the early phase of STZ-diabetes (within
4 wk after STZ injection). Sclerotic glomeruli were evident by
their reduced size and the small number of perfusing glomerular capillary loops (Fig. 6). More importantly, increased
glomerular permeability was readily apparent in the sclerotic
glomeruli (⬃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
Multiphoton microscopy is a state-of-the-art imaging technique which can provide high-resolution images from deep
optical sections of the living tissue, including the kidney (18).
However, multiphoton microscopy has applications far beyond
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Fig. 6. In vivo imaging of glomerular permeability in the diabetic kidney.
Sclerotic glomeruli (arrows) and a hyperfiltering glomerulus (G) are shown.
Note the intense ultrafiltration of the high-molecular-weight (70 kDa) dextranrhodamine B (red) from the plasma into Bowman’s space in the sclerotic, but
not in the hyperfiltering glomerulus.
the generation of superior images: dynamic processes, like the
multiple functions of an intact organ, can be visualized by
real-time imaging in a noninvasive way and quantified. This
study provides further examples of how basic parameters of
renal (patho)physiology can be continuously monitored in the
intact rat kidney.
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 ␮m 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
Innovative Methodology
IN VIVO IMAGING OF BASIC KIDNEY FUNCTIONS
AJP-Renal Physiol • VOL
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.
ACKNOWLEDGMENTS
The authors thank Donald J. Marsh for expert advice and help with the
quantitative analysis of SNGFR.
GRANTS
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-064324 and American Heart Association
Grant EIA 0640056N.
REFERENCES
1. Bird D and Gu M. Two-photon fluorescence endoscopy with a mircooptic scanning head. Opt Lett 28: 1552–1554, 2003.
2. Brown R, Ollerstam A, and Persson AE. Neuronal nitric oxide synthase
inhibition sensitizes the tubuloglomerular feedback mechanism after volume expansion. Kidney Int 65: 1349 –1356, 2004.
3. Burg M, Grantham J, Abramow M, and Orloff J. Preparation and study
of fragments of single rabbit nephrons. Am J Physiol 210: 1293–1298,
1966.
4. Castellino P, Shohat J, and DeFronzo RA. Hyperfiltration and diabetic
nephropathy: is it the beginning? Or is it the end? Semin Nephrol 10:
228 –241, 1990.
5. Castrop H, Lorenz JN, Hansen PB, Friis U, Mizel D, Oppermann M,
Jensen BL, Briggs J, Skøtt O, and Schnermann J. Contribution of the
basolateral isoform of the Na-K-2Cl⫺ cotransporter (NKCC1/BSC2) to
renin secretion. Am J Physiol Renal Physiol 289: F1185–F1192, 2005.
6. Chaigneau E, Oheim M, Audinat E, and Charpak S. Two-photon
imaging of capillary blood flow in olfactory bulb glomeruli. Proc Natl
Acad Sci USA 100: 13081–13086, 2003.
7. Chou CL and Marsh DJ. Measurement of flow rate in rat proximal
tubules with a nonobstructing optical method. Am J Physiol Renal Fluid
Electrolyte Physiol 253: F366 –F371, 1987.
8. Chu J, Chu S, and Montrose MH. Apical Na⫹/H⫹ exchange near the
base of mouse colonic crypts. Am J Physiol Cell Physiol 283: C358 –C372,
2002.
9. Dunn KW, Sandoval RM, Kelly KJ, Dagher PC, Tanner GA, Atkinson SJ, Bacallao RL, and Molitoris BA. Functional studies of the kidney
of living animals using multicolor two-photon microscopy. Am J Physiol
Cell Physiol 283: C905–C916, 2002.
10. Holstein-Rathlou NH. Synchronization of proximal intratubular pressure
oscillations: evidence for interaction between nephrons. Pflügers Arch
408: 438 – 443, 1987.
11. Holstein-Rathlou NH and Leyssac PP. TGF-mediated oscillations in
proximal intratubular pressure: differences between spontaneously hypertensive rats and Wistar-Kyoto rats. Acta Physiol Scand 126: 333–339,
1986.
12. Kaplan MR, Plotkin MD, Brown D, Hebert SC, and Delpire E.
Expression of the mouse Na-K-2Cl cotransporter, mBSC2, in the terminal
inner medullary collecting duct, the glomerular and extraglomerular mes-
291 • AUGUST 2006 •
www.ajprenal.org
Downloaded from http://ajprenal.physiology.org/ by 10.220.32.246 on June 16, 2017
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, fluorescencetagged dextran in the circulating plasma often remains neatly
F501
Innovative Methodology
F502
13.
14.
15.
16.
17.
19.
20.
21.
22.
23.
angium, and the glomerular afferent arteriole. J Clin Invest 98: 723–730,
1996.
Kleinfeld D, Mitra PP, Helmchen F, and Denk W. Fluctuations and
stimulus-induced changes in blood flow observed in individual capillaries
in layers 2 through 4 of rat neocortex. Proc Natl Acad Sci USA 95:
15741–15746, 1998.
Layton AT, Moore LC, and Layton HE. Multistability in tubuloglomerular feedback and spectral complexity in spontaneously hypertensive
rats. Am J Physiol Renal Physiol. In press.
Leyssac PP and Baumbach L. An oscillating intratubular pressure
response to alterations in Henle loop flow in the rat kidney. Acta Physiol
Scand 117: 415– 419, 1983.
Lorenz JN and Gruenstein E. A simple nonradioactive method for
evaluating single-nephron filtration rate using FITC-inulin. Am J Physiol
Renal Physiol 276: F172–F177, 1999.
Marsh DJ, Sosnovtseva OV, Chon KH, and Holstein-Rathlou NH.
Nonlinear interactions in renal blood flow regulation. Am J Physiol Regul
Integr Comp Physiol 288: R1143–R1159, 2005.
Molitoris BA and Sandoval RM. Intravital multiphoton microscopy of
dynamic renal processes. Am J Physiol Renal Physiol 288: F1084 –F1089,
2005.
Oppermann M, Castrop H, Briggs J, and Schnermann J. Influence of
furosemide on renal hemodynamics in mice (Abstract). J Am Soc Nephrol
16: 393A, 2005.
Peti-Peterdi J, Morishima S, Bell PD, and Okada Y. Two-photon
excitation fluorescence imaging of the living juxtaglomerular apparatus.
Am J Physiol Renal Physiol 283: F197–F201, 2002.
Peti-Peterdi J, Fintha A, Fuson AL, Tousson A, and Chow RH.
Real-time imaging of renin release in vitro. Am J Physiol Renal Physiol
287: F329 –F335, 2004.
Peti-Peterdi J. Multiphoton imaging of renal tissues in vitro. Am J Physiol
Renal Physiol 288: F1079 –F1083, 2005.
Sandoval RM, Kennedy MD, Low PS, and Molitoris BA. Uptake and
trafficking of fluorescent conjugates of folic acid in intact kidney deter-
AJP-Renal Physiol • VOL
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
mined using intravital two-photon microscopy. Am J Physiol Cell Physiol
287: C517–C526, 2004.
Sands JM. Micropuncture: unlocking the secrets of renal function. Am J
Physiol Renal Physiol 287: F866 –F867, 2004.
Schnermann J and Briggs J. Function of the juxtaglomerular apparatus:
local control of glomerular hemodynamics. In: The Kidney, edited by
Seldin DW and Giebisch G. New York: Raven, 1985, p. 669 – 697.
Tanner GA, Sandoval RM, and Dunn KW. Two-photon in vivo microscopy of sulfonefluorescein secretion in normal and cystic rat kidneys.
Am J Physiol Renal Physiol 286: F152–F160, 2004.
Tanner GA, Sandoval RM, Molitoris BA, Bamburg JR, and Ashworth
SL. Micropuncture gene delivery and intravital two-photon visualization
of protein expression in rat kidney. Am J Physiol Renal Physiol 289:
F638 –F643, 2005.
Thomson SC, Deng A, Komine N, Hammes JS, Blantz RC, and
Gabbai FB. Early diabetes as a model for testing the regulation of
juxtaglomerular NOS I. Am J Physiol Renal Physiol 287: F732–F738,
2004.
Wearn JT and Richards AN. Observations on the composition of
glomerular urine, with particular reference to the problem of reabsorption
in the renal tubules. Am J Physiol 71: 209 –227, 1924.
Wolf G, Chen S, and Ziyadeh FN. From the periphery of the glomerular
capillary wall toward the center of disease: podocyte injury comes of age
in diabetic nephropathy. Diabetes 54: 1626 –1634, 2005.
Yip KP, Holstein-Rathlou NH, and Marsh DJ. Chaos in blood flow
control in genetic and renovascular hypertensive rats. Am J Physiol Renal
Fluid Electrolyte Physiol 261: F400 –F408, 1991.
Yu W, Sandoval RM, and Molitoris BA. Quantitative intravital microscopy using a Generalized Polarity concept for kidney studies. Am J
Physiol Cell Physiol 289: C1197–C1208, 2005.
Zipfel WR, Williams RM, and Webb WW. Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol 11: 1369 –
1377, 2003.
291 • AUGUST 2006 •
www.ajprenal.org
Downloaded from http://ajprenal.physiology.org/ by 10.220.32.246 on June 16, 2017
18.
IN VIVO IMAGING OF BASIC KIDNEY FUNCTIONS