Evidence for the presence of smooth muscle a-actin
within pericytes of the renal medulla
FRANK PARK,1 DAVID L. MATTSON,1 LOU A. ROBERTS,2 AND ALLEN W. COWLEY, JR.1
of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226;
and 2Department of Biological Sciences, University of Texas-El Paso, El Paso, Texas 79968
1Department
microdissection; reverse transcription-polymerase chain reaction; Western blot analysis; immunohistochemistry; descending vasa recta; renal medullary blood flow
renal medulla has been shown to be
altered independently of the blood flow to the renal
cortex (2, 3). Interestingly, the sole blood supply to the
renal medulla is through the descending vasa recta
capillaries, which are derived entirely from the deep
cortical postglomerular efferent arterioles (14). Descending vasa recta resemble capillaries because they are not
surrounded by vascular smooth muscle cells but instead are surrounded by perivascular elements known
as pericytes (10, 14). Pericytes are believed to be
contractile in nature because they are found to possess
the dense bodies and myofilaments that are necessary
components of contraction (7). Recently, Pallone and
associates have demonstrated that in vitro perfusion of
rat outer medullary descending vasa recta is capable of
vasomodulation when hormonally stimulated by angiotensin II (19) and adenosine (22). This indicates that
these capillaries are capable of functional vasomodulation, at least at the level of the outer medulla, yet there
is little information regarding the physical characteristics of the descending vasa recta pericytical myofila-
BLOOD FLOW TO THE
R1742
ments. The physiological function of pericytes could be
of considerable consequence by acting to modulate
blood flow within the renal medulla, a region not only
important in the concentration of urine but also now
shown to be important in the regulation of sodium
excretion and the long-term control of arterial blood
pressure (2, 3).
Recently, smooth muscle a-actin has been found to be
a useful marker for smooth muscle differentiation (23),
and smooth muscle a-actin has been proposed as a tool
to distinguish pericytes from endothelial cells and
fibroblasts (9) in the bovine retinal microcirculation.
Smooth muscle a-actin has been localized specifically to
the vascular smooth muscle cells and precapillary
pericytes in vivo in the rat renal cortex (1, 4) and rat
mesangial cells in vitro (4). At present, it is not clear
whether smooth muscle a-actin is found within the
renal medulla, specifically surrounding the descending
vasa recta.
This study was therefore designed to determine the
presence of smooth muscle a-actin mRNA and protein
within the pericytes of the renal medulla, using reverse
transcription-polymerase chain reaction (RT-PCR) and
fluorescent immunohistochemistry, respectively.
METHODS
Experimental animals. Tissue for the microdissection of
the descending vasa recta and RT-PCR was obtained from
adult, male Sprague-Dawley rats (200–275 g; Sasco, Madison, WI). Tissue from adult, male Sprague-Dawley rats
(400–600 g) was used for the fluorescent immunohistochemistry. All animals were fed a standard pellet diet (Purina Mills,
St. Louis, MO) and ad libitum water to drink. All protocols
were approved by the Institutional Animal Care and Use
Committee of the Medical College of Wisconsin.
Microdissection of outer medullary descending vasa recta.
Outer medullary descending vasa recta (OMDVR) were microdissected as we have described previously (20). In brief, rats
were first treated with furosemide (5 mg/kg ip). Thirty
minutes later they were anesthetized with ketamine (50
mg/kg) and acepromazine (5 mg/kg) administered intramuscularly. A polyethylene catheter (PE-90) was inserted into the
abdominal aorta distal to the left renal artery, and the left
kidney was selectively perfused with 15-ml dissection solution prewarmed to 37°C followed by perfusion with 0.6 ml of
2.5% latex-coated brown-dyed microparticles (,1–4 µm in
diameter; Polysciences, Warrington, PA). After the perfusion,
the kidney was removed and cut coronally, and the renal
medulla was excised. The medullary tissue was cut into
smaller pieces and placed into the dissection solution containing 1 mg/ml collagenase (CLS 2, 192 U/mg; Worthington
Biochemical, Freehold, NJ) at 37°C for 35–40 min. The
kidney slices were rinsed with dissection solution and then
placed under a Zeiss M3Z stereomicroscope (magnification
316–100) for dissection. Four separate kidneys were used in
0363-6119/97 $5.00 Copyright r 1997 the American Physiological Society
Downloaded from http://ajpregu.physiology.org/ by 10.220.32.246 on June 17, 2017
Park, Frank, David L. Mattson, Lou A. Roberts, and
Allen W. Cowley, Jr. Evidence for the presence of smooth
muscle a-actin within pericytes of the renal medulla. Am. J.
Physiol. 273 (Regulatory Integrative Comp. Physiol. 42):
R1742–R1748, 1997.—This study was designed to determine
whether smooth muscle a-actin mRNA and smooth muscle
a-actin contractile protein elements were present within the
renal medullary pericytes. Extraction of total RNA from
microdissected outer medullary descending vasa recta allowed for the detection of smooth muscle a-actin mRNA
expression using reverse transcription-polymerase chain reaction (RT-PCR). Expression of smooth muscle a-actin was
specific to the descending vasa recta and not a result of
tubular contamination because RT-PCR amplification of the
vasopressin V2 receptor, which is a specific tubular marker,
did not occur. To determine the exact cell type(s) that translate the mRNA into protein, we performed immunohistochemistry on the renal outer and inner medulla using a monoclonal
smooth muscle a-actin antibody, whose specificity was determined by immunoblot analysis. Smooth muscle a-actin protein was found selectively within the pericytes surrounding
the descending vasa recta from the outer and inner medullary
tissue sections. This study demonstrates that the pericytes
alone that surround the descending vasa recta within the
outer and inner medulla contain smooth muscle a-actin
mRNA and protein and are therefore the site of the contractile elements that could play a vasomodulatory role in the
control of renal medullary blood flow and its distribution
within the renal medulla.
SMOOTH MUSCLE a-ACTIN IN THE RENAL MEDULLARY PERICYTES
Table 1. Nucleotide sequence of the RT-PCR primers
Name
Sequence
Size
Location
Smooth muscle
a-Actin
58-GAT CAC CAT CGG GAA 389 bp 789–810
TGA ACG C-38 (sense)
58-CTT AGA AGC ATT TGC
1,156–1,177
GGT GGA C-38 (antisense)
58-CTG GAA GGT AGA TAG
1,097–1,108
AGA AGC C-38 (internal)
b-Actin
58-AAC CGC GAG AAG ATG 350 bp 383–413
ACC CAG ATC ATG
TTT-38 (sense)
58-AGC AGC CGT GGC CAT
703–733
CTC TTG CTC GAA
CTG-38 (antisense)
Vasopressin
58-ATG GTG GGC ATG TAT 461 bp 399–427
V2 receptor
GCC TCC TCC TAC
ATG-38 (sense)
58-AGT GTC ATC CTC ACG
835–859
GTC TTG GCC A-38 (antisense)
RT-PCR, reverse transcription-polymerase chain reaction.
1 µl was used for b-actin. The reaction mixture was first
denatured at 94°C for 5 min and then cycled for 35 cycles
between 94°C (denaturation) for 1 min, 64°C (annealing) for 1
min, and 72°C (extension) for 1 min. Samples were incubated
for an additional 7 min at 72°C after the completion of the
final cycle.
PCR product analysis. From each of the PCR reactions,
10-µl aliquots were size-fractionated by electrophoresis on a
1.6% agarose gel. After electrophoresis and ethidium bromide
staining, DNA bands were visualized with an ultraviolet
transilluminator. To verify the authenticity of the PCR products, the gels were denatured, neutralized, and blotted onto a
nylon membrane (Micron Separations) for Southern blot
analysis. The DNA was cross-linked to the membrane with
ultraviolet light and hybridized with an internal oligonucleotide, which was labeled 38 with a fluorescein nucleotide
(Amersham UK). The final blot wash was 13 saline-sodium
citrate-0.1% sodium dodecyl sulfate (SDS) at 58°C. To further
authenticate the specificity of the PCR products, PCR sequencing using the dideoxynucleotide method was performed (Amersham, Arlington Heights, IL).
Protein isolation. The kidneys from anesthetized male
Sprague-Dawley rats were cut in a coronal section to isolate
the outer medulla. The outer medullary tissue was homogenized in (in mM) 5 K2HPO4, 5 KH2PO4, 250 sucrose, pH 7.7,
0.1 EDTA, 0.1 phenylmethylsulfonyl fluoride, 2 µg/µl leupeptin, and 5 µg/µl pepstatin. The homogenate was centrifuged
at 1,000 g for 10 min to remove any incompletely homogenized membrane fragments and nuclei, and the supernatant
was centrifuged at 16,000 g for 20 min. The 16,000-g supernatant was removed for determination of protein concentration
using the Coomassie method (Pierce, Rockford, IL), and the
proteins were frozen at 280°C.
Electrophoresis and immunoblotting of membranes. Sample buffer [2% SDS, 100 mM Tris · HCl, pH 6.8, 5% bmercaptoethanol, 12% (vol/vol) glycerol, and 0.02% (wt/vol)
Bromphenol blue] was added to the protein sample, and the
mixture was heated to 100°C for 5 min. Five micrograms of
outer medullary protein were loaded onto the gel and then
size separated by electrophoresis through a 12% SDSpolyacrylamide gel. The proteins were transferred onto a
nitrocellulose membrane (Bio-Rad), and the membranes were
blocked with 15% nonfat dried milk in blotting solution
overnight at 4°C. The blotting solution contained (in mM) 137
NaCl, 20 Tris · HCl, pH 7.4, and 0.08% Tween 20. The membranes were incubated for 30 min with the monoclonal
smooth muscle a-actin primary antibody (1:2,500; Sigma, St.
Louis, MO) at room temperature. The membranes were
washed in several changes of blotting buffer, and then incubated for 30 min with secondary antibody (goat anti-rabbit
immunoglobulin G, 1:1,000; Bio-Rad). The membranes were
washed, and the protein bands were detected by chemiluminescence (WesternView, Transduction Laboratories) on X-ray
film.
Immunohistochemistry of the renal outer and inner medulla. Kidneys were perfusion-cleared with Tyrode medium
and subsequently perfused with india ink in Tyrode medium.
The kidney tissue was hemisected and cut into wedges
containing the cortex and outer and inner medulla. The
wedges were cryoprotected by treatment with increasing
concentrations of sucrose (10, 20, and 30%) in phosphatebuffered saline (PBS), pH 7.4. The tissue was then frozen on
dry ice in OCT embedding medium (Scientific Products,
McGaw Park, IL) and stored in liquid nitrogen. Frozen
sections (50 µm thick) were made using a cryostat set at
220°C, and sections were placed on glass slides ringed with
rubber cement. The sections were thawed for 5 min, fixed for
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the microdissection of the OMDVR, in which each kidney
supplied ,20–25 individual OMDVR from the inner stripe of
several outer medullary vasa recta bundles. Vessel lengths
were measured by an optical micrometer, and ,12 mm of
OMDVR were isolated per kidney dissection. The dissected
vessels were transferred to another dish to wash off any
contaminating debris and then transferred to a thin-walled
ultracentrifuge tube containing 100 µl TRIZOL reagent
(GIBCO-BRL, Gaithersburg, MD) for permeabilization of the
isolated tissue.
RNA isolation and RT. Total RNA was extracted from the
isolated OMDVR (,12 mm in total length) as previously
described by adding 50 µl chloroform. The aqueous phase was
removed after centrifugation, and the RNA was precipitated
with isopropanol (70 µl). The RNA pellet was then washed
with 75% ethanol, allowed to dry at room temperature, and
resuspended in deoxyribonuclease (DNase) solution containing 1 U RQ1 ribonuclease-free DNase and 20 U RNasin
(Promega Biotech, Madison, WI) to remove any genomic DNA
contamination. The RNA was then reextracted as described
above, and the RNA pellet was allowed to dry at room
temperature for 5 min. RT was performed on the total RNA
using 0.5 µg oligo(dT)15—18 (Promega Biotech) as previously
described (19). cDNA was then synthesized at 37°C for 60
min, and the reaction was stopped by heating to 95°C for 5
min.
Preparation of oligonucleotide primers. All nucleotide primers were purchased from Operon Technologies (Alameda,
CA). Oligonucleotide primers are shown in Table 1 and were
chosen from the published cDNA sequences of rat smooth
muscle a-actin (12), human b-actin (7), and the vasopressin
V2 receptor (V2R) (10). The primers for b-actin and smooth
muscle a-actin primers were designed to span at least one
intron. The V2R primers are found on the same exon.
PCR. All PCR reactions were performed in a total volume of
50 µl in the presence of (in mM) 0.2 dNTP, 10 1,4dithiothreitol, 50 KCl, 1.0 MgCl2, and 10 tris(hydroxymethyl)aminomethane (Tris) · HCl, pH 8.3, as well as 50 pmol of each
primer, 2.5 U AmpliTaq polymerase (Perkin-Elmer Cetus,
Norwalk, CT). Mineral oil was layered on top of each sample
to prevent evaporation of the liquid. All primer cDNA amplifications were optimum under these standard conditions.
Seven-microliter aliquots of the RT reactions were used in the
amplification for both smooth muscle a-actin and V2R, whereas
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SMOOTH MUSCLE a-ACTIN IN THE RENAL MEDULLARY PERICYTES
hand sliced, and then processed as described for cryosectioned tissue. The thick slices were mounted in PPD on large
glass slides for study.
RESULTS
10 min in 95% ethanol, rinsed in PBS, and then rinsed in PBS
containing Triton X-100 (Sigma). They were incubated with
the smooth muscle a-actin antibody (clone 1A4; Sigma) at a
concentration of 1:50 in PBS at room temperature for 1 h. The
sections were washed in PBS containing bovine serum albumin and were subsequently incubated in secondary antibody
(goat anti-mouse labeled with rhodamine, 1:50 in PBS; Boehringer-Mannheim) for 1 h at room temperature. The sections
were washed in PBS and then mounted in 50% glycerol
containing 1% p-phenylenediamine (PPD). The sections were
studied with a Nikon Optiphot fluorescence microscope.
Kidneys that were used for antibody staining of 0.5- to
1-mm-thick slices were cleared as described above and fixed
by perfusion of 4% ammonium molybdate in distilled water.
The kidney was hemisected and cut into wedges as above,
Fig. 2. Ethidium bromide-stained agarose gels of reverse transcription-polymerase chain reaction (RT-PCR) products from
outer medullary tissue (lane 2; positive
control) and microdissected outer medullary descending vasa recta (lane 3) for
smooth muscle a-actin (A), b-actin (C),
and vasopressin V2 receptor (D). Corresponding Southern blot analyses for
smooth muscle a-actin (B) verify the specificity of the amplified RT-PCR products.
Expected size of the PCR product was 389
bp for smooth muscle a-actin, 461 bp for
the V2 receptor, and 350 bp for b-actin; the
positions of the DNA size markers (in bp)
are shown at left. Negative control samples
are shown in lane 4 (PCR amplification of
sterile water), lane 5 (PCR amplification
of dissection solution), and lane 6 (PCR
amplification of DNase-treated RNA).
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Fig. 1. Micrograph of a microdissected outer medullary descending
vasa recta capillary from a vascular bundle (magnification 3200).
Smooth muscle a-actin mRNA expression in OMDVR.
OMDVR from the outer medullary vasa recta bundles
were microdissected and total RNA was extracted to
determine whether these isolated vessels expressed
smooth muscle a-actin mRNA. In each experiment, a
pool of descending vasa recta totaling ,12 mm in
length was obtained by microdissection from each
individual kidney. OMDVR were individually microdissected and inspected under 3100 magnification to
achieve the purest microvessel preparation. Descending vasa recta were identified by their very thin (,20
µm in diameter) and ‘‘bumpy’’ wall appearance, which
was related to the presence of the cell bodies of the
pericyte. Figure 1 illustrates the clean microdissection
of an isolated OMDVR from the vascular bundles with
no evident contaminating debris from surrounding
structures, particularly renal tubules. This lack of
debris is an important and stringent criterion, which
was implemented to accept these vessels for RNA
extraction and RT-PCR. Figure 2 shows the RT-PCR
results of the total RNA from isolated OMDVR for
smooth muscle a-actin (Fig. 2A), b-actin (Fig. 2C), and
V2R (Fig. 2D). In each microdissection and RT-PCR
that was performed (n 5 4), outer medullary tissue
RNA was used as a positive control (lane 2) and lane 3
shows that microdissected OMDVR express smooth
muscle a-actin. In all of the kidneys used for microdissection, the presence of smooth muscle a-actin was
clearly evident (n 5 4). The specificity of the RT-PCR
products were shown with Southern blot analysis using
an internal oligonucleotide probe for smooth muscle
a-actin (Fig. 2B) and by subsequent sequencing of the
RT-PCR products (which showed that the PCR product
was 100% homologous compared with smooth muscle
a-actin sequences found within GenBank).
RT-PCR of the V2R mRNA, which we have described
previously (19), was used as a negative control to
ascertain tubular contamination (i.e., medullary thick
SMOOTH MUSCLE a-ACTIN IN THE RENAL MEDULLARY PERICYTES
ascending limb of Henle and/or outer medullary collecting ducts), which can occur during the microdissection
and RNA extraction of OMDVR. The absence of V2R
mRNA within the OMDVR (Fig. 2D, lane 3) verified the
purity of these microvessels and lack of tubular contamination.
The results from RT-PCR demonstrate that the mRNA
for smooth muscle a-actin is found within microdissected OMDVR (lane 3). Bands were not detected in
any of the negative control samples, which included, in
lane 4, PCR amplification of sterile water; in lane 5,
PCR amplification of dissection solution; and in lane 6,
PCR amplification of DNase-treated RNA (V2R). The
negative control for the PCR amplification of DNasetreated RNA was necessary for only the V2R primers
because these primers did not span introns like those
for smooth muscle a-actin and b-actin.
Specificity of smooth muscle a-actin antibody by
immunoblot analysis using outer medullary protein.
Initial experiments were performed to determine the
specificity of the monoclonal smooth muscle a-actin
antibody in protein preparations from the outer medulla. Figure 3 shows that the monoclonal antibody
specifically recognizes a 47-kDa protein, which is the
expected size of smooth muscle a-actin, in outer medullary protein isolated from two different rat outer medullas (lanes 1 and 2).
Fluorescent immunohistochemistry of the outer and
inner medulla. The specificity of the monoclonal antibody allowed for the immunolocalization of smooth
muscle a-actin within sections of the outer and inner
medulla of the kidney. Figure 4 demonstrates the
fluorescence of smooth muscle a-actin surrounding
descending vasa recta from the outer (Fig. 4, A-D) and
inner medulla (Fig. 4E) of hand-sliced tissue. Figure 4A
(magnification 3200) and Fig. 4B (magnification
31,000) show an efferent arteriole branching into a
vasa recta bundle near the outer-inner stripe of the
outer medulla. As shown by the small arrow, the
immunofluorescence was localized to the cell body of
the pericyte and its extensions. Figure 4, C and D,
demonstrates the smooth muscle a-actin within descending vasa recta in the inner stripe of the outer
medulla. Figure 4E shows inner medullary descending
vasa recta also exhibiting abundant smooth muscle
a-actin.
Interestingly, as shown in Fig. 5, which was from a
cryostat section of renal medullary tissue, the immunofluorescence of smooth muscle a-actin was found to
continue deep within the inner medulla and disappeared only near the tip of the papilla. Because this
kidney was only partially filled with india ink, a better
visualization of several outer medullary vascular
bundles was achieved. Smooth muscle a-actin fluorescence was clearly present in the pericytic cell bodies
and its tentacle-like extensions, which surrounded the
descending vasa recta. Because of the specificity of the
smooth muscle a-actin antibody in the pericytes, descending vasa recta could be immunofluorescently distinguished from other vascular, tubular, and interstitial structures. Moreover, it is important to note that
ascending vasa recta are not associated with pericytes,
and thus no immunofluorescence was observed. No
immunofluorescence was detected in about the distal
one-third of the inner medulla. It is evident from Figs. 4
and 5 that the pericytes surrounding the outer and
inner medullary vasa recta capillaries are circumferentially well positioned to alter microvascular diameters
when stimulated to contract.
DISCUSSION
The application of molecular biological techniques
has allowed for the determination of gene transcription
and subsequent protein translation within minute tissue samples such as microdissected tubules and blood
vessels as was required for the present studies. RT-PCR
is an extremely sensitive technique that allows for the
amplification of reverse-transcribed cDNA from nearly
single copies of mRNA transcripts, but it does not
provide information regarding the cellular origin of the
mRNA. Moreover, the presence of the mRNA does not
assure that protein is being translated from the mRNA
within the specific cells of interest. For this reason, in
the present study, fluorescent immunohistochemistry
using antibodies specific for smooth muscle a-actin was
used to localize specific cell type(s) that translated the
smooth muscle a-actin mRNA into proteins. By coupling RT-PCR with immunohistochemistry, the results
of this study determined the localization of the renal
medullary gene expression of smooth muscle a-actin
and further discriminated that the cellular site of
protein translation was within the vasa recta pericytes.
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Fig. 3. Specificity of smooth muscle a-actin was shown by Western
blot analysis using outer medullary protein (5 µg/lane; lanes 1 and 2).
Lanes 1 and 2 are protein isolated from two different rat outer
medullas (OM). Protein standards are indicated at left. Molecular
weight of smooth muscle a-actin is 47 kDa (as indicated by arrow on
right).
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SMOOTH MUSCLE a-ACTIN IN THE RENAL MEDULLARY PERICYTES
The importance of contractile smooth muscle a-actin
found within renal medullary pericytes relates to the
role of these cells in determining the vascular diameter
of the vasa recta and consequently the regulation of
renal medullary blood flow. Contractile elements, specifically smooth muscle a-actin, have been found within
pericytes that surround the extrarenal microcirculation of the bovine retina and rat mesentery (9, 18). In
the kidney, there is definitive evidence that vascular
smooth muscle cells in the renal cortex of rats possess
smooth muscle a-actin (1, 4), but there is little known
about its existence within the renal medulla. Histologi-
cal studies by Moffatt (14) demonstrated that renal
medullary pericytes surrounded descending vasa recta
and that these pericytes contained myofibrils that were
similar to those found within the vascular smooth
muscle cells, but the type of myofibril was not determined. Carey et al. (1) determined the expression of
smooth muscle a-actin within the renal medulla of
Wistar rats but concluded that its existence was developmentally regulated because these investigators observed smooth muscle a-actin within the renal medulla
in very young (15 day old and younger) rats. The results
of the present study conclusively demonstrate the
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Fig. 4. Fluorescently labeled anti-smooth muscle a-actin-stained pericytes along the proximal vasa recta bundle
near the outer-inner stripe border of the outer medulla (A and B) in hand-sliced tissue. Arrows in A (magnification
3200) and B (magnification 31,000) point to the same pericytic cell body surrounding a descending vasa recta
capillary. C and D show smooth muscle a-actin immunofluorescence of descending vasa recta from different vasa
recta bundles in the inner stripe of the outer medulla. E shows immunofluorescence for smooth muscle a-actin on
inner medullary descending vasa recta. C-E are at 31,000 magnification. Solid bar 5 60 µm in A and 12 µm in B-E.
SMOOTH MUSCLE a-ACTIN IN THE RENAL MEDULLARY PERICYTES
R1747
presence of smooth muscle a-actin in the pericytes of
medullary descending vasa recta in adult rats. The
divergence in the results may be attributed to strain
differences (Wistar vs. Sprague Dawley) but is more
likely due to differences in the methodological approaches used in the detection of smooth muscle a-actin
(fluorescence vs. diaminobenzidine staining).
Pericytes within the kidney are heterogeneous in
nature (21) and may participate in several ways to
modify microvessel function. Morphologically, pericytes
of the peritubular capillaries in the renal cortex are
aligned along the longitudinal axis of the vessels (4),
whereas the pericytes surrounding the descending vasa
recta in the renal medulla are aligned longitudinally
and circumferentially to the vessels as seen in Fig. 4.
Consistent with these morphological characteristics,
Murphy and Wagner (15) found that cultured pericytes
were capable of contracting in two ways: first, tangentially (longitudinally) to the vessel, which would be
expected to increase the permeability of the capillaries,
or, second, circumferentially, which would modulate
vessel diameter and modulate blood flow. This would be
consistent with observations by Pallone and associates
(19, 22), who have seen reductions of descending vasa
recta diameters in isolated perfused vasa recta capillaries from the rat outer medulla superfused with hormonal vasoconstrictors.
A potentially important physiological role for the
presence of smooth muscle a-actin within inner medullary descending vasa recta is related to the ability of
these contractile elements to differentially regulate
blood flow distribution within the renal medulla. In
vivo functional studies in our laboratory (12, 16, 17)
using implanted laser-Doppler flowmetry techniques
have now shown that selective infusion of vasoactive
compounds into the renal medullary interstitial space
can lead to preferential modulation of renal medullary
blood flow. Franchini and Cowley (6) have also observed
that 48-h water restriction in conscious SpragueDawley rats preferentially reduced inner medullary
blood flow without altering the blood flow to the outer
medulla. In addition, videomicroscopic studies by Fenoy
and Roman (5) showed that volume expansion with
intravenous saline infusion was associated with an
increase in the number of functionally perfused vasa
recta capillaries within the renal medulla, suggesting a
mechanism for recruitment of previously unperfused
descending vasa recta. The observations from these
studies and the visually observed reductions of OMDVR
diameter with angiotensin and vasopressin (19, 24)
indicate that the pericytes play a pivotal role in the
modulation of renal medullary blood flow.
In conclusion, this study has shown that smooth
muscle a-actin mRNA is found in microdissected descending vasa recta capillaries and that the protein is
translated and found specifically within the pericytes
surrounding these vasa recta capillaries within the
outer and inner medulla.
The authors would like to thank Meredith M. Skelton for her
critical reading of this manuscript.
F. Park was supported by a Wisconsin Affiliate American Heart
Predoctoral Fellowship (96-F-PRE-15). This study was supported by
National Heart, Lung, and Blood Institute Grant HL-49219.
Address for reprint requests: F. Park, Dept. of Physiology, Medical
College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI,
53226.
Received 21 May 1997; accepted in final form 14 August 1997.
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Fig. 5. Immunofluorescence for smooth
muscle a-actin (left) and light microscopy (right) of inner medullary tissue
intermittently filled with india ink in
cryostat-sectioned tissue. Note the specific immunofluorescence of the descending vasa recta from the inner
stripe of the outer medulla down to the
inner medulla. No immunofluorescence
was observed in ascending vasa recta.
Black arrow shows approximate border
between the inner stripe of the outer
medulla and the inner medulla. Specific fluorescence for smooth muscle aactin descended to near the papillary
tip of the kidney section. Magnification
3100; solid bar 5 120 µm.
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SMOOTH MUSCLE a-ACTIN IN THE RENAL MEDULLARY PERICYTES
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