ORIGINAL ARTICLE

Rho-kinase Contributes to Pressure-induced Constriction of
Renal Microvessels

Koichiro Homma,1,2 Koichi Hayashi,1 Shu Wakino,1 Hirobumi Tokuyama,1 Takeshi Kanda,1 Satoru Tatematsu,1Kazuhiro Hasegawa,1 Seitaro Fujishima,2 Shingo Hori, 2 Takao Saruta1 and Hiroshi Itoh1
1Departments of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan
2Emergency Medicine, School of Medicine, Keio University, Tokyo, Japan(Received for publication on February 8, 2013)(Revised for publication on October 1, 2013)(Accepted for publication on October 16, 2013)(Published online in advance on January 16, 2014)

Renal afferent arterioles (AFF) regulate glomerular capillary pressure through two main mechanisms:
the myogenic response (MYO) and tubuloglomerular feedback (TGF). Because Rho-kinase and nitricoxide synthase (NOS) are established factors that modulate vascular tone, we examined the role ofthese factors in pressure-induced AFF tone in Wistar-Kyoto rats and in spontaneously hypertensive
rats (SHR) using an intravital CCD camera. Elevated renal perfusion pressure elicited marked AFF
constriction that was partially inhibited by gadolinium, furosemide and fasudil, which inhibit MYO,TGF and Rho-kinase, respectively; however, this AFF constriction was completely blocked by combined
treatment with fasudil+gadolinium or fasudil+furosemide. S-methyl-l-thiocitrulline (SMTC) partiallyreversed the fasudil-induced inhibition of TGF-mediated, but not that of MYO-mediated, AFF constriction. In SHR, the pressure-induced AFF response was enhanced, and MYO- and TGF-induced constriction were exaggerated. In the presence of gadolinium, SMTC partially mitigated the fasudil-induced inhibition of TGF-mediated AFF constriction. Immunoblot analyses demonstrated that both Rho-kinase
activity and neuronal NOS were augmented in SHR kidneys. In conclusion, Rho-kinase contributes to
MYO- and TGF-mediated AFF responses, and these responses are enhanced in SHR. Furthermore,neuronal NOS-induced nitric oxide modulates the TGF mechanism. This mechanism constitutes a target for Rho-kinase in TGF-mediated AFF constriction. (doi: 10.2302/kjm.2013-0001-OA ; Keio J Med
63 (1) : 1–12, March 2014)

Keywords: Rho-kinase, myogenic response, tubuloglomerular feedback, nNOS, intravital CCD
cameradisease.4,5 Additionally, in the renal microcirculation, themyogenic response and the tubuloglomerular feedback
mechanism (TGF) constitute the two main mechanisms
responsible for renal autoregulation and are mediated by
active adjustments of vascular smooth muscle tone, primarily in the afferent arterioles (AFF).6 Recently, Rho/
Rho-kinase was found to be responsible for the generation of renal arteriolar tone, and Y-27632, a Rho/Rhokinase inhibitor, was found to decrease basal renal arteriolar tone and impair the myogenic response of AFF in

Introduction
Rho/Rho-kinase plays an important role in the regulation of vascular tone by enhancing calcium sensitivity.1
A growing body of evidence has indicated that activated Rho/Rho-kinase in vascular beds is responsible forpathophysiological conditions, including hypertension 2
and coronary vasospasm.3 Furthermore, the Rho/Rhokinase pathway has been demonstrated to contribute to
the development of renal injury in various models of renal

Reprint requests to: Koichi Hayashi, MD, PhD, Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi,
Shinjuku, Tokyo 160-8582, Japan, E-mail: khayashi@z8.keio.jp
Copyright © 2014 by The Keio Journal of Medicine
1

Homma K, et al: Rho-kinase and Renal Microcirculation

2hydronephrotic kidneys (in which AFF tone is regulated
mainly by the myogenic response of renal arterioles) bothin vivo7 and in vitro8. Nevertheless, the role of Rho/Rhokinase in the TGF mechanism remains to be determined.
Nitric oxide (NO) is produced by three isoforms of NO
synthase (NOS): endothelial NOS (eNOS), inducible NOS
and neuronal NOS (nNOS). Although it has been postulated that in the kidney eNOS predominantly synthesizes
NO, recent studies have shown that nNOS is abundantly
expressed in macula densa cells, Bowman’s capsules,
cells of the thick ascending limb and endothelial cellsof renal microvessels.9 Furthermore, nNOS-derived NO
plays an important role in counteracting the modulationof TGF-mediated AFF constriction.10 Recently, an interaction between NO and the Rho/Rho-kinase pathway
was demonstrated. Shin et al.11 reported that Rho-kinase
negatively regulates eNOS activity in the acutely ischemic brain. Furthermore, the Rho/Rho-kinase signaling
pathway is amplified in kidneys from eNOS-deficientmice.12 In this regard, it has been demonstrated that the
Rho-kinase pathway participates in adenosine-induced
contraction of renal arterioles.13 Because adenosine has
been established as a factor mediating TGF, there exists
a complex relationship among Rho-kinase, NOS and adenosine in the renal microcirculatory milieu. Althoughthese interactions have attracted much interest, no studies
have examined the mutual actions among these components.
In the present study, we examined the role of Rho/
Rho-kinase in mediating pressure-induced changes inrenal arteriolar tone in rat kidneys using an intravitalneedle-type CCD camera technique to directly visualize the renal microcirculation. Furthermore, the interaction between Rho/Rho-kinase and nNOS in mediatingpressure-induced AFF constriction was also investigated.
Finally, we evaluated these responses in kidneys from
Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) to clarify whether hypertension modifies
these responses.
Materials and Methods
Intravital, pencil-lens-probe, charge-coupled-device
videomicroscopy
The intravital videomicroscope used in the present
study was a modification of a needle-lens probe previously detailed elsewhere.14 Images on the CCD were converted into black-and-white video signals every 33 ms
and recorded on videocassette tape. The spatial resolution of this system was determined by the use of a UnitedStates Air Force 1951 test target to be 0.9 μm for ×600
magnification on a 15-inch video monitor.

Animal preparation
All experimental procedures were conducted in accordance with the guidelines of the Animal Care Committeeof Keio University. Nine- to eleven-week-old male WKY
and SHR (Charles-River Japan, Kanagawa, Japan) were
fed a standard laboratory chow. The animals were anesthetized with pentobarbital sodium (50 mg/kg intraperitoneal injection) and the left kidney was exposed via aflank incision and isolated from the surrounding tissues.
The tip of the CCD camera probe was introduced into
the renal cortex, and AFF diameters were assessed. The
left carotid and femoral arteries were cannulated (PE50,
Becton Dickinson, Franklin Lakes, NJ) to measure arterial blood pressure with the use of a pressure transducer(TP4009, Nihon Koden, Tokyo, Japan). The pressure
measured at the carotid artery was assumed to be the renal perfusion pressure (RPP).15
Measurement of microvascular diameter
After the surgical procedure and instrumentation, thepencil-lens probe was introduced into the superficial cortex.14 The probe was moved inside slowly to obtain clear
images of glomeruli and the adjoining afferent arterioles
using a three-dimensional micromanipulator. After determination of the microvascular area for observation,
the probe was pulled back in several increments of 10 µm
to prevent direct compression of the glomerular network.
AFF were distinguished by the direction of the blood
flow. Video images were continuously recorded on videocassette tape.
The sequential images of renal microvessels were analyzed by a computer (840AV; Apple Computer, Cupertino, CA, USA) using a freeze-frame modality. The density in gray-scale mode was digitized to an arbitrary unit
along the scanning line across the vessel. The differencein the gray scale between the peak value and the background noise level was divided into quarters. The position with the density value a quarter higher than the noiselevel was identified as the inner wall of the vessel, as described previously.16 The diameters were determined byaveraging at least five measurements during the plateauof the response.
Immunoblotting of Rho-kinase and nNOS
Renal cortical tissues were lysed and sonicated in solubilization buffer. Immunoblotting was performed as described previously,17,18 using specific antibodies against
phospho-MYPT1 (Upstate Biochemistry, Lake Placid,
NY, USA) and nNOS (Transduction Laboratories, Lexington, KY, USA). Immunoreactive bands were detectedusing an ECL detection kit (Amersham Biosciences, Uppsala, Sweden).

Keio J Med 2014; 63 (1): 1–12

3

Experimental protocols

Statistical analysis

Protocol 1: Effect of elevated RPP on renal microvascular tone
One hour was allowed after stabilization of renal hemodynamics. After observation of the basal AFF diameters
in WKY and SHR, RPP was elevated by clamping the celiac artery and the superior mesenteric artery. Then, RPP
was further elevated by clamping the descending aorta at
the peripheral level of renal arteries. The AFF diameters
were measured at each RPP.

Data are expressed as the mean ± SEM. Data were analyzed by two-way analysis of variance (ANOVA), followed by Bonferroni’s multiple-comparison post hoc test.
P < 0.05 was defined as statistically significant.

Protocol 2: Effect of gadolinium and furosemide onpressure-induced changes in AFF tone
The roles of TGF and the myogenic response in mediating pressure-induced AFF vasoconstriction in vivo
was evaluated using gadolinium (Gd) and furosemide
in WKY and SHR. Gd is a relatively selective blocker
of mechanosensitive cation channels and thereby abolishes myogenic constriction.19,20 In contrast, loop diuretics such as furosemide cause significant diuresis andnatriuresis by inhibiting the Na-K-2Cl cotransporter inthe thick ascending limb of Henle’s loop, including themacula densa, and induce vasodilation through inhibitionof the TGF mechanism.21 Initially, Gd was infused intravenously (5 or 10 mg/kg; Sigma Chemicals, St. Louis,
MO, USA). After 5 min, the effect of the stepwise RPP
elevation on AFF diameter was assessed. Similarly, AFF
responses to elevated RPP were examined 5 min after
initiation of intravenous infusion of furosemide (10 or
16 µg/kg/min; Sigma Chemicals).

Figure 1A shows representative images of AFF responses to elevated RPP in WKY. On elevation of RPP,
AFF manifested marked constriction, whereas no constrictor response was observed in the efferent arteriole. The responses of AFF to pressure are summarized
in Figure 1B. Elevation of RPP from 78 ± 6 to 106 ± 6mmHg tended to reduce AFF diameter (from 12.6 ± 0.5
μm to 11.9 ± 0.5 μm, n =10, P > 0.20), and elevation to 140± 8 mmHg elicited 17.5 ± 2.0% constriction (from 12.6 ±0.5 μm to 10.6 ± 0.4 μm, n =10, P < 0.05).

Protocol 3: Role of Rho-kinase in pressure-induced AFF
tone
The participation of Rho-kinase in pressure-induced
AFF constriction was evaluated in WKY and SHR. Initially, fasudil (Asahi Kasei, Tokyo, Japan) was infused
intravenously at 0.3 or 0.6 mg/kg for 5 min. Thereafter,
the pressure-induced changes in AFF diameter were assessed in WKY and SHR.
We further evaluated the role of nNOS-associated nitricoxide in Rho-kinase-mediated AFF constriction. S-methyl-l-thiocitrulline (SMTC; Sigma Chemicals), an nNOSselective inhibitor,22 was infused at a rate of 20 µg/kg/
min. Then, the inhibitory action of fasudil (0.6 mg/kg)
on pressure-induced AFF constriction was evaluated in
WKY and SHR.
To further clarify whether SMTC-associated NO inhibition affected myogenic or TGF inhibition by fasudil, we
examined the effect of fasudil on pressure-induced AFF
tone under pretreatment with SMTC (20 µg/kg/min) andGd (10 mg/kg) or furosemide (16 µg/kg/min). Thus, fasudil was injected intravenously in the presence of Gd
or furosemide, and the effect of fasudil on the pressureinduced changes in renal arteriolar tone was evaluated.

Results
Protocol 1: Effect of elevated RPP on renal microvascular tone

Protocol 2: Effect of Gd and furosemide on pressureinduced changes in AFF tone
Neither Gd (5 and 10 mg/kg) nor furosemide (10 and
16 µg/kg/min) had any effect on the basal AFF diameter in WKY (data not shown). The addition of Gd (5 and
10 mg/kg) blunted the pressure-induced changes in AFF
diameter in a dose-dependent manner in WKY, (n =10,
Fig. 2). Similarly, furosemide (10 and 16 µg/kg/min) significantly inhibited the pressure-induced constriction of
AFF (n =10). Furthermore, combined treatment with Gd(10 mg/kg) and furosemide (16 µg/kg/min) completely
abolished this response (n =10).
Protocol 3: Role of Rho-kinase in pressure-induced
AFF toneAs shown in Figure 3A, the administration of fasudil
dilated AFF in a dose-dependent manner at baseline RPP
in WKY (baseline, 11.5 ± 0.5 μm; 0.3 mg/kg, 12.2 ± 0.5
μm; 0.6 mg/kg, 13.3 ± 0.5 μm; P < 0.05; n =10).
In the presence of fasudil (0.6 mg/kg), pressure-induced
AFF constriction was blunted, with only 1.1 ± 1.1% (P <0.05 vs. control) and 4.3 ± 0.8% decrements (P < 0.01 vs.
control) observed at 110 ± 8 and 143 ± 6 mmHg, respectively (Fig. 3B, lower).
The ability of fasudil to inhibit the TGF-mediated andmyogenic AFF tone was evaluated in WKY. In the presence of Gd (10 mg/kg), fasudil (0.6 mg/kg) abolished the
AFF constriction induced by elevated RPP (1.1 ± 0.8% vs.
9.5 ± 1.2% decrease in diameter) (Fig. 4). Similarly, in the

4Homma K, et al: Rho-kinase and Renal Microcirculation

Fig. 1 Visualization of renal microcirculation in vivo and responses to elevated renal perfusion pressure in Wistar-Kyoto rats (WKY).
An intravital CCD camera allowed direct visualization of afferent arteriolar responses to elevated renal perfusion pressure (RPP) (A).
Stepwise elevation in RPP by clamping the superior mesenteric artery (SMA)+celiac artery and SMA+celiac artery+aorta elicited
RPP-dependent constriction of afferent arterioles (AFF) (B). EFF; efferent arterioles. *P<0.05 vs. baseline.

Fig. 2 Myogenic and tubuloglomerular feedback (TGF)-mediated constriction of the afferent arteriole in WKY.
Gadolinium and furosemide blunted pressure-induced afferent arteriolar constriction in a dose-dependent manner. Pretreatment withboth gadolinium and furosemide completely abolished the response. *P <0.05 vs. control, **P <0.01 vs. control.

Keio J Med 2014; 63 (1): 1–12

5

Fig. 3 Effect of fasudil on basal and pressure-induced tone of afferent arterioles in WKY.
Under basal conditions, fasudil dilated afferent arterioles in a dose-dependent manner (A). Fasudil markedly blunted the pressureinduced vasoconstriction of afferent arterioles (B). RPP was elevated stepwise by clamping the SMA+celiac artery and SMA+celiac
artery+aorta. *P <0.05 vs. baseline.

Fig. 4 Effect of fasudil on pressure-induced constriction of the afferent arteriole in the presence of gadolinium or furosemide in WKY.
Fasudil (0.6 mg/kg) completely abolished the pressure-induced constriction of afferent arterioles treated with gadolinium (Gd, 10 mg/kg) or furosemide (Furo, 16 µg/kg/min).

6presence of furosemide (16 µg/kg/min), fasudil (0.6 mg/kg) abolished the RPP-induced AFF constriction (1.2 ±
2.0% increase vs. 7.8 ± 0.8% decrease in diameter).
Next, the role of nNOS in fasudil inhibition of pressure-induced AFF constriction was examined in WKY.
SMTC (20 µg/kg/min), an nNOS-selective inhibitor, had
no effect on basal AFF tone but enhanced pressure-induced constriction (23.2 ± 1.6% vs. 17.5 ± 2.0% decreasein diameter, P < 0.05, n =10, Fig. 5A). When the effectof SMTC on fasudil-induced inhibition was assessed,
SMTC markedly restored the AFF response to elevatedHomma K, et al: Rho-kinase and Renal Microcirculation

RPP (12.0 ± 1.6% vs. 4.3 ± 0.8% decrease in diameter, P< 0.05, n =10).
We further examined whether the effects of SMTC on
fasudil-induced inhibition were mediated by a myogenic
or TGF mechanism. In the presence of Gd (10 mg/kg),
fasudil (0.6 mg/kg) completely abolished the AFF constriction induced by elevated RPP (1.1 ± 0.8% decreasein diameter, Fig. 5B). The addition of SMTC (20 µg/
kg/min) reversed the fasudil-induced inhibition of RPPinduced AFF constriction (9.1 ± 0.6%, P < 0.05) to thelevel observed in the presence of Gd (i.e., 9.5 ± 1.2%, P

Fig. 5 Role of neuronal nitric oxide synthase (nNOS) in myogenic and tubuloglomerular feedback-induced afferent arteriolar tone in
WKY.
S-methyl-l-thiocitrulline (SMTC), an nNOS-selective inhibitor, augmented pressure-induced afferent arteriolar constriction. Fasudil(0.6 mg/kg)-induced inhibitory effects on the pressure-constricted afferent arterioles were substantially mitigated by SMTC (A).
SMTC potently reversed the fasudil-induced inhibition in the presence of gadolinium (Gd, 10 mg/kg; B), but not in the presence of
furosemide (Furo, 16 µg/kg/min; C). *P <0.05 vs. control

Keio J Med 2014; 63 (1): 1–12

7

Fig. 6 Pressure-induced constrictor responses of the afferent arteriole in SHR and WKY.
The same magnitude of elevation in renal perfusion pressure caused a larger vasoconstrictor response in SHR than in WKY (A). Whenthe inhibitory action of gadolinium or furosemide on pressure-induced constriction was evaluated, the ability of gadolinium to inhibit
afferent arteriolar constriction tended to be greater in SHR than in WKY (B). Similarly, the inhibition of pressure-induced constriction
by furosemide was greater in SHR (C). *P <0.05 vs. control, **P <0.01 vs. control.

> 0.5). Simultaneous treatment with furosemide (16 µg/
kg/min) and fasudil (0.6 mg/kg) abolished RPP-induced
AFF constriction (1.2 ± 2.0% increase in diameter, Fig.
5C). In this setting, however, SMTC failed to reverse thefasudil-induced inhibition of AFF vasoconstriction.
Protocol 4: RPP-induced constriction in SHR
The basal AFF diameter in SHR (10.0 ± 0.4 μm, n =10)
was slightly less than that in WKY (12.6 ± 0.5 μm, P <0.05). AFF of SHR exhibited 12.2 ± 2.0% and 24.9 ± 2.3%
decreases in diameter (n =10, P < 0.01) in response to elevated RPP (Fig. 6A). It is of note that the AFF constriction in SHR was greater than that in WKY in response to
the same magnitude of elevated RPP.
The ability of Gd and furosemide to inhibit elevated
RPP-induced AFF constriction was compared in WKY
and SHR. Both Gd and furosemide inhibited RPP-induced AFF vasoconstriction in a dose-dependent manner
in WKY and SHR (Figs. 6B and C). Of note, the inhibito-ry action of Gd tended to be greater in SHR than in WKY,
and the ability of furosemide to inhibit RPP-induced AFF
vasoconstriction was actually enhanced in SHR.
The administration of fasudil at baseline RPP dilated
AFF from 9.3 ± 0.5 µm to 11.0 ± 0.5 µm (0.3 mg/kg; P< 0.05, n =10) and 12.5 ± 0.6 μm (0.6 mg/kg; P < 0.01, n=10) in SHR, which corresponded to 18.3 ± 3.3% and 23.7
± 4.0% vasodilation, respectively (Fig. 7A); the vasodilator responses to fasudil were greater in SHR than in
WKY (P < 0.05).
Fasudil markedly inhibited the pressure-induced constriction of AFF in SHR (control, 24.9 ± 2.3% decrease;fasudil, 7.3 ± 1.5% decrease; Fig. 7B, black bars). It isof note that fasudil eliminated the difference in RPPinduced AFF vasoconstriction between WKY and SHR.
Pretreatment with SMTC enhanced the pressure-induced AFF constriction in both SHR and WKY (Fig.
7C; see also Fig. 7B, control). Furthermore, SMTC restored the fasudil-induced impairment in the AFF constrictor response to pressure more greatly in SHR than

8

Homma K, et al: Rho-kinase and Renal Microcirculation

Fig. 7 Effects of fasudil on basal and pressure-induced afferent arteriolar constriction in SHR.
The vasodilator effect of fasudil on basal afferent arteriolar tone was larger in SHR than in WKY (A). Although the pressure-induced
constrictor response was greater in SHR, fasudil (Fas, 0.6 mg/kg) prevented this response to the same extent in SHR and WKY (B). In
contrast, pretreatment with SMTC mitigated the inhibitory action of fasudil (Fas) during pressure-induced AFF constriction, and thismitigation effect was more pronounced in SHR (C). SMTC restored the pressure-induced AFF constriction that was almost completely
inhibited by gadolinium (Gd, 10 mg/kg)+Fas, and this restorative action was greater in SHR (D). White bars, WKY; black bars, SHR. #P <0.05 vs. baseline, ## P <0.01 vs. baseline. †† P <0.01 vs. control. * P <0.05 vs. SMTC. § P <0.05 vs. Gd+Fas, §§ P <0.01 vs. Gd+Fas.

in WKY (Fig. 7C; see also Fig. 7B, Fas). An enhanced
AFF responses in SHR was also apparent when the effect of SMTC was examined in the presence of Gd; thefasudil+Gd-induced inhibition of pressure-induced AFF
constriction was identical in WKY and SHR, whereas in
the presence of SMTC, a greater constrictor response wasobserved in SHR (14.3 ± 0.9%, n =10) than in WKY (8.6
± 1.1%, n =10) (Fig. 7D). These observations suggest thatfasudil-induced inhibition of pressure-induced AFF constriction is mediated in part by nNOS-induced NO andthe resulting TGF inhibition, and this mechanism is exaggerated in SHR. In the presence of furosemide, however,
SMTC failed to reverse the inhibition by fasudil of the
AFF constrictor response to pressure (data not shown).
Figure 8A illustrates the comparison of Rho-kinase

activity (as assessed by phosphorylation of MYPT) in
kidneys from WKY and SHR. It was clearly demonstrated that Rho-kinase activity was greater in SHR than in
WKY (n =3). Similarly, intrarenal nNOS was upregulated
in SHR relative to WKY (n =3, Fig. 8B).
Discussion
In the present study, we confirmed that both myogenic
and TGF-mediated AFF constriction can be visualized in
an in vivo, in situ and relatively intact setting (Figs. 1, 2).
Furthermore, fasudil was found to elicit dose-dependent
relaxation of the basal and pressure-constricted tone ofAFF (Fig. 3). These findings indicate that the Rho-kinase
pathway is involved not only in the generation of basal

Keio J Med 2014; 63 (1): 1–12

9Fig. 8 Comparison of Rho-kinase activity and nNOS expression in WKY and SHR.
Renal Rho-kinase activity, as assessed by phosphorylation of MYPT-1, was enhanced in SHR (A). Similarly, nNOS expression was
greater in SHR than in WKY (B). # P <0.01 vs. WKY.

tone but also in the development of pressure-induced tonein the renal microcirculation. Moreover, further inhibition of pressure-induced constriction by fasudil during
the inhibition of myogenic and TGF-induced tone by Gd
and furosemide, respectively, suggests that Rho-kinase
makes a substantial contribution to both of these vasoconstrictor mechanisms (Fig. 4). In addition, we recently
demonstrated that Y-27632 (a selective Rho-kinase inhibitor) prevents pressure-induced AFF constriction in theisolated perfused hydronephrotic rat kidney8 in which the
TGF-mediated AFF constrictor mechanism is absent.23
This finding suggests a crucial role for Rho-kinase in the
myogenic mechanism of pressure-induced AFF constriction. The present study further indicates the involvementof Rho-kinase in TGF-mediated AFF tone in addition to
its role in myogenic tone. Thus, our current study clearlysupports the concept that Rho-kinase constitutes a determinant of both myogenic and TGF-induced AFF tone.
It is well established that the TGF mechanism involves
multiple components that affect AFF tone in a positiveor negative manner. Among these components, NO isassumed to be a modulator of TGF that counters TGFmediated AFF constriction.10,22 Indeed, we and other laboratories previously reported that elevated RPP elicited
increases in renal NO levels.24,25 Immunohistochemical
studies have revealed the presence of nNOS in the kidney, with expression primarily localized to the cells of
the macula densa,26 and selective nNOS inhibition causes
a 32–42% reduction in renal cortical and medullary NO
levels in normotensive rats.27 These observations suggestthat nNOS-dependent NO production constitutes a significant portion of the total NO generation within the kidney. Collectively, our study is consistent with the premisethat TGF is influenced by nNOS-induced NO.
The present study shows that the inhibition of nNOS
by SMTC enhances pressure-induced AFF constriction(Fig. 5A). Furthermore, the ability of SMTC to restorepressure-induced AFF constriction is preserved during
the treatment with fasudil (12.0 ± 1.6% vs. 4.3 ± 0.8%,
P < 0.01). It follows, therefore, that the inhibition by fa-

10sudil of pressure-induced AFF constriction is modulated
in part by the vasodilator action of nNOS-generated NO.
We carried out further experiments to delineate the roleof Rho-kinase in TGF-mediated AFF constriction. Thus,in the presence of Gd, in which TGF is assumed to bea main constrictor mechanism, fasudil completely abolished pressure-induced AFF constriction. In this setting,
SMTC pronouncedly reversed this response (Fig. 5B).
These observations strongly suggest that the inhibitionof TGF by fasudil is mediated largely by the activationof nNOS. It is known that Rho-kinase exerts multifaceted effects that are necessary to maintain various cellular functions. In addition to the Ca-sensitizing action of
Rho-kinase in vascular smooth muscle cells, Rho-kinase
activation causes the suppression of eNOS activity.11,28
Furthermore, our finding that SMTC reverses the fasudilinduced suppression of TGF lends support to the hypothesis that Rho-kinase modulates nNOS activity and thesubsequent production of NO.
In contrast, in the presence of furosemide, SMTC
fails to affect the fasudil-induced inhibition of pressureinduced AFF constriction (Fig. 5C). This observationsuggests that nNOS-induced NO appears to exert only
modest action on myogenic AFF constriction. In thisregard, by using nNOS-knockout mice, Dautzenberg etal.29 showed that nNOS-generated NO had no significant
effect on the myogenic response. Several studies haveexamined the effect of NO on the myogenic response of
the renal microcirculation. An in vivo study revealed that
NO inhibition fails to alter the autoregulatory efficiencyof renal blood flow, although basal blood flow was reduced.30 These results suggest that NO does not impair
the myogenic AFF response. Similarly, nitro-l-arginine
methyl ester (a L-NAME) has no effect on the myogenic
AFF responses in the in vivo hydronephrotic kidney31
or fails to enhance the efficiency of myogenic AFF constriction.32 Finally, nitroprusside, cyclic GMP, and atrial
natriuretic peptide, which shares its intracellular signaltransduction pathway with NO, failed to inhibit myogenic
AFF constriction.33 In contrast, recent studies have demonstrated that TGF-mediated NOS activation modifies
myogenic autoregulation34 and the AFF response to pressure.35 Because the dilator action of NO is mediated inpart by protein kinase G and the inhibition of Rho A,36
this mechanism might explain how NO could modulate
the myogenic AFF response. A schematic illustrating thepossible roles of Rho-kinase in the myogenic response
and in TGF and the association between Rho-kinase andnNOS is shown in Figure 9.
Several studies have shown that the renal microvascular response to pressure is altered in hypertension. Thus,
myogenic constriction was reported to be enhanced in
AFF from SHR.37 Furthermore, Welch et al.38 showedthat TGF is augmented in AFF from SHR. In the present study, we demonstrated that the AFF of SHR kidneys
manifest exaggerated responses to elevated pressure (Fig.
6A). Furthermore, both TGF and myogenic components

Homma K, et al: Rho-kinase and Renal Microcirculation

are enhanced in SHR compared to those in WKY (Figs.
6B and C). These in vivo findings are therefore consistent
with the previous in vitro observations described above
and support the concept that an enhanced AFF constrictor response to pressure protects against high systemic
blood pressure in hypertensive animals.
The mechanisms underlying the augmented AFF constrictor response to pressure in hypertension remain undetermined. One possible mechanism is enhanced Rhokinase activity in hypertensive animals. Uehata et al.2
demonstrated that Y-27632, a Rho-kinase inhibitor, elicited an exaggerated depressor action when administeredin hypertensive animals. In the present study, we examined the role of Rho-kinase in renal microvascular tone
in SHR and observed an enhanced vasodilator effect offasudil on the AFF (Fig. 7A). Furthermore, fasudil abolished the exaggerated pressure-induced AFF constrictor
response observed in SHR (Fig. 7B). These observations
suggesting enhanced Rho-kinase activity in SHR kidneys were confirmed by the immunoblotting of MYPT-1(Fig. 8A). It follows, therefore, that Rho-kinase activity
constitutes an important determinant of the enhanced
pressure-induced AFF constriction in SHR.
The role of nNOS in mediating the renal microvascular action of Rho-kinase in hypertensive animals remains
unclear. In contrast to the effect of fasudil on pressureinduced AFF constriction (Fig. 7B), pretreatment with
SMTC mitigated the inhibitory action of fasudil on pressure-induced AFF constriction, and this effect was more
pronounced in SHR (Fig. 7C). Thus, SMTC restored thedifference in pressure response between WKY and SHR.
Furthermore, in the presence of Gd, i.e., when TGF is
predominantly mediating the pressure-induced AFF constriction, the reversal by SMTC of fasudil-induced inhibition of the pressure response is exaggerated in SHR(Fig. 7D). These observations raise the possibility thataugmented Rho-kinase in SHR suppresses nNOS activity
and NO production, which subsequently enhances TGF
signaling. Finally, renal nNOS expression is upregulated
in SHR (Fig. 8B), which may serve to counter suppressed
NO activity or bioavailability.39
In conclusion, we demonstrated that the Rho/Rhokinase pathway is involved in the pressure-induced toneof the renal microcirculation. This novel mechanism exerts its action through several vasomotor components
within the renal microcirculation, including the TGF andmyogenic tone, and is further affected at least in part bynNOS-associated TGF modulation. Finally, the exaggerated pressure-induced tone in hypertensive animals
is attributable to the enhanced activity of Rho-kinase. It
awaits further investigations to determine whether exaggerated Rho-kinase activity protects against glomerular
hypertension or contributes to the progression of renal
injury in hypertension.

Keio J Med 2014; 63 (1): 1–12

11

Fig. 9 Schematic illustrating the role of Rho-kinase in myogenic and tubuloglomerular feedback-mediated afferent arteriolar vasoconstriction.
Elevated renal perfusion pressure enhances both myogenic and TGF mechanisms and causes afferent arteriolar vasoconstriction. Rhokinase mediates or modulates the myogenic mechanism. TGF is under the negative control of nNOS-mediated nitric oxide (NO). Rhokinase is assumed to suppress nNOS activity and NO production, leading to augmentation of TGF-mediated afferent arteriolar vasoconstriction. Gadolinium (Gd) is a selective inhibitor for mechanosensitive cation channels that mediate myogenic vasoconstriction.
Furosemide inhibits the Na/K/2Cl cotransporter in the macula densa that plays an important role in TGF. S-methyl-l-thiocitrulline(SMTC) is a blocker of nNOS activity and suppresses NO production.

References
1. Narumiya S: The small GTPase Rho: cellular functions and signaltransduction. J Biochem 1996; 120: 215–228. [Medline] [CrossRef]
2. Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita
T, Tamakawa H, Yamagami K, Inui J, Maekawa M, Narumiya
S: Calcium sensitization of smooth muscle mediated by a Rhoassociated protein kinase in hypertension. Nature 1997; 389:
990–994. [Medline] [CrossRef]
3. Shimokawa H: Rho-kinase as a novel therapeutic target in treatment of cardiovascular diseases. J Cardiovasc Pharmacol 2002;
39: 319–327. [Medline] [CrossRef]
4. Kanda T, Wakino S, Hayashi K, Homma K, Ozawa Y, Saruta T:
Effect of fasudil on Rho-kinase and nephropathy in subtotally nephrectomized spontaneously hypertensive rats. Kidney Int 2003;
64: 2009–2019. [Medline] [CrossRef]
5. Nishikimi T, Akimoto K, Wang X, Mori Y, Tadokoro K, Ishikawa
Y, Shimokawa H, Ono H, Matsuoka H: Fasudil, a Rho-kinase inhibitor, attenuates glomerulosclerosis in Dahl salt-sensitive rats. J
Hypertens 2004; 22: 1787–1796. [Medline] [CrossRef]
6. Hayashi K, Epstein M, Saruta T: Altered myogenic responsiveness of the renal microvasculature in experimental hypertension.
J Hypertens 1996; 14: 1387–1401. [Medline] [CrossRef]
7. Cavarape A, Endlich N, Assaloni R, Bartoli E, Steinhausen M,
Parekh N, Endlich K: Rho-kinase inhibition blunts renal vasoconstriction induced by distinct signaling pathways in vivo. J Am Soc
Nephrol 2003; 14: 37–45. [Medline] [CrossRef]

8. Nakamura A, Hayashi K, Ozawa Y, Fujiwara K, Okubo K, Kanda
T, Wakino S, Saruta T: Vessel- and vasoconstrictor-dependentrole Rho/Rho-kinase in renal microvascular tone. J Vasc Res
2003; 40: 244–251. [Medline] [CrossRef]
9. Mount PF, Power DA: Nitric oxide in the kidney: functions andregulation of synthesis. Acta Physiol (Oxf) 2006; 187: 433–446.
[Medline] [CrossRef]
10. Ren YL, Garvin JL, Carretero OA: Role of macula densa nitricoxide and cGMP in the regulation of tubuloglomerular feedback.
Kidney Int 2000; 58: 2053–2060. [Medline] [CrossRef]
11. Shin HK, Salomone S, Potts EM, Lee SW, Millican E, Noma K,
Huang PL, Boas DA, Liao JK, Moskowitz MA, Ayata C: Rhokinase inhibition acutely augments blood flow in focal cerebral
ischemia via endothelial mechanisms. J Cereb Blood Flow Metab.
2006 Oct 11.
12. Williams J, Bogwu J, Oyekan A: The role of the RhoA/Rho-kinase signaling pathway in renal vascular reactivity in endothelial
nitric oxide synthase null mice. J Hypertens 2006; 24: 1429–1436.
[Medline] [CrossRef]
13. Lai EY, Martinka P, Fahling M, Mrowka R, Steege A, Gericke
A, Sendeski M, Persson PB, Persson AE, Patzak A: Adenosine
restores angiotensin II-induced contractions by receptor-independent enhancement of calcium sensitivity in renal arterioles. Circ
Res 2006; 99: 1117–1124 Epub 2006 Oct 12. [Medline] [CrossRef]
14. Matsuda H, Hayashi K, Arakawa K, Naito M, Kubota E, Honda
M, Matsumoto A, Suzuki H, Yamamoto T, Kajiya F, Saruta T:
Zonal heterogeneity in action of angiotensin-converting enzyme

12
inhibitor on renal microcirculation: role of intrarenal bradykinin.
J Am Soc Nephrol 1999; 10: 2272–2282. [Medline]
15. Cowley AW Jr, Mattson DL, Lu S, Roman RJ: The renal medulla
and hypertension. Hypertension 1995; 25: 663–673. [Medline][CrossRef]
16. Yada T, Hiramatsu O, Kimura A, Goto M, Ogasawara Y, Tsujioka
K, Yamamori S, Ohno K, Hosaka H, Kajiya F: In vivo observationof subendocardial microvessels of the beating porcine heart using
a needle-probe videomicroscope with a CCD camera. Circ Res
1993; 72: 939–946. [Medline] [CrossRef]
17. Wakino S, Hayashi K, Kanda T, Tatematsu S, Homma K, Yoshioka K, Takamatsu I, Saruta T: Peroxisome proliferator-activatedreceptor gamma ligands inhibit Rho/Rho kinase pathway by inducing protein tyrosine phosphatase SHP-2. Circ Res 2004; 95:
e45–e55. [Medline] [CrossRef]
18. Wakino S, Hayashi K, Tatematsu S, Hasegawa K, Takamatsu I,
Kanda T, Homma K, Yoshioka K, Sugano N, Saruta T: Pioglitazone lowers systemic asymmetric dimethylarginine by inducing dimethylarginine dimethylaminohydrolase in rats. Hypertens
Res 2005; 28: 255–262. [Medline] [CrossRef]
19. Yang XC, Sachs F: Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions. Science 1989; 243:
1068–1071. [Medline] [CrossRef]
20. Wellner M.C., Isenberg G: Stretch-activated non-selective cation channels in urinary bladder myocytes: importance for pacemaker potentials and myogenic response. Non-Selective Cation
Channels: Pharmacology, Physiology and Biophysics, pp 93–99.
Birkhauser Verlag Basal, Switzerland.
21. Wright FS, Schnermann J: Interference with feedback control ofglomerular filtration rate by furosemide, triflocin, and cyanide. J
Clin Invest 1974; 53: 1695–1708. [Medline] [CrossRef]
22. Ichihara A, Navar LG: Neuronal NOS contributes to biphasic
autoregulatory response during enhanced TGF activity. Am J
Physiol 1999; 277: F113–F120. [Medline]
23. Steinhausen M, Blum M, Fleming JT, Holz FG, Parekh N, Wiegman DL: Visualization of renal autoregulation in the split hydronephrotic kidney of rats. Kidney Int 1989; 35: 1151–1160. [Medline] [CrossRef]
24. Fujiwara K, Hayashi K, Matsuda H, Kubota E, Honda M, Ozawa
Y, Saruta T: Altered pressure-natriuresis in obese Zucker rats.
Hypertension 1999; 33: 1470–1475. [Medline] [CrossRef]
25. Majid DS, Omoro SA, Chin SY, Navar LG: Intrarenal nitric oxide
activity and pressure natriuresis in anesthetized dogs. Hypertension 1998; 32: 266–272. [Medline] [CrossRef]
26. Tojo A, Welch WJ, Bremer V, Kimoto M, Kimura K, Omata M,
Ogawa T, Vallance P, Wilcox CS: Colocalization of demethylating
enzymes and NOS and functional effects of methylarginines in
rat kidney. Kidney Int 1997; 52: 1593–1601. [Medline] [CrossRef]
27. Mattson DL, Higgins DJ: Influence of dietary sodium intake onrenal medullary nitric oxide synthase. Hypertension 1996; 27:
688–692. [Medline] [CrossRef]

Homma K, et al: Rho-kinase and Renal Microcirculation
28. Shiga N, Hirano K, Hirano M, Nishimura J, Nawata H, Kanaide
H: Long-term inhibition of RhoA attenuates vascular contractility
by enhancing endothelial NO production in an intact rabbit mesenteric artery. Circ Res 2005; 96: 1014–1021. [Medline] [CrossRef]
29. Dautzenberg M, Keilhoff G, Just A: Modulation of the myogenic
response in renal blood flow autoregulation by NO depends onendothelial nitric oxide synthase (eNOS), but not neuronal or inducible NOS. J Physiol 2011; 589: 4731–4744. [Medline]
30. Racasan S, Joles JA, Boer P, Koomans HA, Braam B: NO dependency of RBF and autoregulation in the spontaneously hypertensive rat. Am J Physiol Renal Physiol 2003; 285: F105–F112.
[Medline]
31. Hoffend J, Cavarape A, Endlich K, Steinhausen M: influence ofendothelium-derived relaxing factor on renal microvessels andpressure-dependent vasodilation. Am J Physiol 1993; 265: F285–
F292. [Medline]
32. Hayashi K, Suzuki H, Saruta T: Nitric oxide modulates but does
not impair myogenic vasoconstriction of the afferent arteriolein spontaneously hypertensive rats. Studies in the isolated perfused hydronephrotic kidney. Hypertension 1995; 25: 1212–1219.
[Medline] [CrossRef]
33. Hayashi K, Epstein M, Loutzenhiser R: Determinants of renal
actions of atrial natriuretic peptide. Lack of effect of atrial natriuretic peptide on pressure-induced vasoconstriction. Circ Res
1990; 67: 1–10. [Medline] [CrossRef]
34. Just A, Arendshorst WJ: Nitric oxide blunts myogenic autoregulation in rat renal but not skeletal muscle circulation via tubuloglomerular feedback. J Physiol 2005; 569: 959–974. [Medline][CrossRef]
35. Shi Y, Wang X, Chon KH, Cupples WA: Tubuloglomerular feedback-dependent modulation of renal myogenic autoregulation by
nitric oxide. Am J Physiol Regul Integr Comp Physiol 2006; 290:
R982–R991. [Medline]
36. Sauzeau V, Le Jeune H, Cario-Toumaniantz C, Smolenski A,
Lohmann SM, Bertoglio J, Chardin P, Pacaud P, Loirand G:
Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascularsmooth muscle. J Biol Chem 2000; 275: 21722–21729. [Medline][CrossRef]
37. Ito S, Juncos LA, Carretero OA: Pressure-induced constriction of
the afferent arteriole of spontaneously hypertensive rats. Hypertension 1992; 19(Suppl): II164–II167. [Medline] [CrossRef]
38. Welch WJ, Tojo A, Wilcox CS: Roles of NO and oxygen radicals
in tubuloglomerular feedback in SHR. Am J Physiol Renal Physiol 2000; 278: F769–F776. [Medline]
39. Tojo A, Onozato ML, Fujita T: Role of macula densa neuronal
nitric oxide synthase in renal diseases. Med Mol Morphol 2006;
39: 2–7. [Medline] [CrossRef]