Articles in PresS. Am J Physiol Regul Integr Comp Physiol (July 24, 2013). doi:10.1152/ajpregu.00226.2013
Female SHR have greater blood pressure sensitivity and renal T cell infiltration
following chronic NOS inhibition than males.
Krystal N. Brinson*1, Ahmed A. Elmarakby*2,3, Ashlee J. Tipton1, G. Ryan Crislip1,
Tatsuo Yamamoto4, Babak Baban2, Jennifer C. Sullivan1
Departments of Medicine1, Oral Biology2, and Pharmacology & Toxicology3, Georgia
Regents University, Augusta, GA; Second Department of Medicine4, Numazu City
Hospital, Shizuoka Japan.
* Indicates equal contribution as first author.
Running Head: L-NAME Hypertension in male and female SHR
Manuscript Word Count (including references, figures, legends): 6,979
Abstract Word Count: 246
Number of Tables: 5
Number of Figures: 7
Corresponding Author: Jennifer C. Sullivan
Georgia Regents University
1459 Laney Walker Blvd, CB-2200
Augusta, GA 30912
Phone: 706-721-9796
Fax: 706-721-7661
Email: [email protected]
1
Copyright © 2013 by the American Physiological Society.
Abstract.
Nitric oxide is a critical regulator of blood pressure (BP) and inflammation and female
spontaneously hypertensive rats (SHR) have higher renal nitric oxide bioavailability than
males. We hypothesize that female SHR will have a greater rise in BP and renal T cell
infiltration in response to nitric oxide synthase (NOS) inhibition than males. Both male
and female SHR displayed a dose-dependent increase in BP to the non-specific NOS
inhibitor NG-nitro-L-arginine methyl ester (L-NAME: 2, 5, and 7 mg/kg/day for 4 days
each); however, females exhibited a greater increase in BP than males. Treatment of
male and female SHR with 7 mg/kg/day L-NAME for 2 weeks significantly increased BP
in both sexes; however, prior exposure to L-NAME only increased BP sensitivity to
chronic NOS inhibition in females. L-NAME-induced hypertension increased renal T cell
infiltration and indices of renal injury in both sexes, yet female SHR exhibited greater
increases in Th17 cells and greater decreases in regulatory T cells than males. Chronic
L-NAME was also associated with larger increases in renal cortical adhesion molecule
expression in female SHR.
The use of triple therapy to block L-NAME-mediated
increases in BP attenuated L-NAME-induced increases in renal T cell counts and
normalized adhesion molecule expression in SHR, suggesting that L-NAME-induced
increases in renal T cells were dependent on both increases in BP and NOS inhibition.
Our data suggest that NOS is critical in the ability of SHR, females in particular, to
maintain BP and limit a pro-inflammatory renal T cell profile.
Keywords: nitric oxide synthase, kidney injury, hypertension, sex, inflammation
2
Introduction
Clinical and experimental studies have established that there is a sex difference
in the incidence of hypertension, with males developing high blood pressure (BP) at
younger ages than females (7, 33). The molecular mechanisms responsible for the sex
difference remains unclear, although the nitric oxide (NO)/NO synthase (NOS) pathway
has been implicated. NO is a key regulator of BP (4, 13) and the incidence and
progression of hypertension has been linked with NO deficiencies in experimental
animal models (3, 4). There is also a sex difference in the NO/NOS pathway; NO
bioavailability is greater in females compared to males (11, 14). Therefore, a sex
difference in the NO/NOS system may contribute to sexual dimorphisms in BP.
Spontaneously hypertensive rats (SHR) are a genetic model of hypertension
where females have a slower elevation in BP with age compared to males (27, 33). We
recently reported that female SHR have greater renal NO bioavailability than males
(32).
However, the implication of these findings on the role of NOS in BP control
remains unknown. Chronic NOS inhibition induces hypertension in normotensive male
rats, and exacerbates the progression of hypertension in male SHR (4, 24, 35). There
are conflicting reports in the literature regarding the impact of sex on the response to
chronic NOS inhibition. Normotensive female rats have been reported to be more
sensitive (36), less sensitive (19, 30, 35) or equally sensitive (38) to NOS inhibitioninduced hypertension compared to age-matched males.
T cells play a critical role in the progression of hypertension and renal injury in
male experimental models of hypertension (10, 16), and we recently published that
female SHR have a less pro-inflammatory renal T cell profile than males (34). NO
3
inhibits adhesion molecule expression in vitro and in vivo to decrease vascular
monocyte adhesion (9, 15), and lymphocyte suppression with mycophenolate mofetil
(MMF) attenuates NG-nitro-L-arginine methyl ester (L-NAME)-induced hypertension in
male Sprague-Dawley rats (12, 26). However, it is unknown how chronic NOS inhibition
impacts the renal T cell profile in either sex. Based on the importance of the NOS
pathway in maintaining BP and the greater NO bioavailability in female SHR compared
to males (32), the current study was designed to test the hypothesis that female SHR
are more dependent on NOS to maintain BP compared to males.
We also
hypothesized that greater sensitivity of female SHR to L-NAME-induced hypertension
will be associated with greater elevations in renal T cell infiltration.
Methods
Animals
Male and female SHR (Harlan Laboratories, Indianapolis, IN) were studied in
accordance with the National Institutes of Health Guide for the Care and Use of
Laboratory Animals, and approved and monitored by the Georgia Regents University
IACUC. At 10 weeks of age male and female SHR were implanted with telemetry
devices (Data Sciences, St. Paul, MN) to monitor BP as previously described (31, 33).
Rats were allowed one week recovery and one week of baseline BP recording before
being randomized to receive vehicle (tap water) or the NOS inhibitor L-NAME. Two
experimental approaches were employed to assess the contribution of NO to BP control
in male and female SHR: 1) BP dose response and 2) chronic L-NAME treatment. For
all studies L-NAME was given in drinking water. Rats were individually housed to allow
4
for daily measurements of water intake; all rats were weighed every three days. LNAME was administered according to both weight and water intake to maintain
appropriate dosage in each sex. At the end of all studies rats were anesthetized with
ketamine/xylazine (50 mg/kg and 6 mg/kg i.p., respectively; Phoenix Pharmaceuticals,
St. Joseph, MO), a terminal blood sample was taken and kidneys were isolated.
L-NAME Dose Response
Rats were randomized to receive vehicle (tap water; n=5) or increasing doses of
L-NAME in drinking water: 2, 5 and 7 mg/kg/day (n=5-6; Sigma-Aldrich, St. Louis, MO).
Each dose was given for 4 days. Pilot studies using 0.1, 0.5 and 2 mg/kg/day L-NAME
revealed 2 mg/kg/day L-NAME as the lowest dose to result in a change in BP from
baseline in either sex (data not shown). L-NAME treatment was then suspended for
one week to assess the ability of BP to return to baseline values in both sexes prior to
initiating chronic BP studies.
Chronic L-NAME
7 mg/kg/day (equivalent to ~ 80 mg/L) L-NAME is comparable to commonly
used doses in the literature to induce L-NAME hypertension (5, 24). Two groups of
male and female SHR were treated with L-NAME at 7 mg/kg/day for 14 days (n=7-9).
Group 1: rats from the dose-response study; Group 2: age-matched rats that had not
previously been exposed to L-NAME (L-NAME naïve).
L-NAME naïve rats were
included to assess not only the impact of prior exposure to L-NAME on the chronic BP
responses, but also because BP did not return to baseline values in female SHR
following the L-NAME dose-response studies in Group 1.
Triple Therapy
5
An additional set of male and female SHR were treated with L-NAME at 7
mg/kg/day for 14 days in the presence of triple therapy (TTx: reserpine, hydralazine,
and hydrochlorothiazide (20); n=5) to block L-NAME-induced increases in BP. TTx was
not used to abolish hypertension in SHR; BP in these rats was maintained at levels
comparable to vehicle control SHR.
Renal Health and Histological Analysis
A subset of rats were placed in metabolic cages for 24-hour urine collection
before beginning and at the end of the 7 mg/kg/day L-NAME treatment to measure total
protein (Bradford assay; Bio-Rad, Hercules, CA), albumin and nephrin excretion
(Exocell, Philadelphia, PA).
Kidneys were prepared for histological analysis as
previously described to assess renal injury and slides were examined in a blinded
manner (33).
Histological examination of kidneys was performed following staining
using periodic acid-Schiff (PASH), Masson’s trichrome, and Picro-Sirus Red staining.
Analytical flow cytometry
Whole blood and cell suspensions of kidneys were prepared and phenotypic and
intracellular analyses performed as previously described to determine numbers of CD3+
and CD4+ T cells, regulatory T cells (Tregs; CD3+/CD4+/Foxp3+) or Th17 cells
(CD3+/CD4+/ROR-γ+; all antibodies from BD Biosciences, San Diego, CA) (1, 34).
Antibody specificity was confirmed using isotype controls.
Real-time Polymerase Chain Reaction (RT-PCR)
RNA was isolated from 30-50 mg of renal cortex and medulla from control, LNAME and L-NAME/TTx-treated SHR (n=5-6) using RNeasy Plus Mini kit (all reagents
and primers from Qiagen, Valencia, CA).
A blend of oligonucleotide and random
6
hexanucleotide primers were used for reverse transcription of 1 µg of RNA using
Quantifect Reverse Transcription kit and RT-PCR was performed with QuantiTect
SYBR Green RT-PCR kit. GAPDH was the internal standard and mRNA levels were
expressed relative to control in each sex.
Statistical Analysis
All data are expressed as mean ± SEM. Telemetry data within each sex were
analyzed using repeated measures ANOVA with Greenhouse-Geisser correction.
Telemetry data between sexes and between control and L-NAME treated rats were
compared using T-test. Flow cytometry, adhesion molecule, and urinary excretion data
were compared using one-way ANOVA followed by a Newman-Keul’s post-hoc test.
For all comparisons, differences were statistically significant with p<0.05.
Analyses
were performed using GraphPad Prism Version 5.0 (GraphPad Software Inc., La Jolla,
CA).
Results
Animal Characteristics
Dose-Response: At baseline, male SHR were larger than females (274±6 vs. 177±2 g;
p<0.05) and consumed more water (30±1 vs. 26±2 mL; p<0.05). Over the course of the
study, both sexes gained weight (males: 42±4 g; females: 16±2 g). There was not a
difference in weight gain in vehicle vs. L-NAME treated rats. Water consumption did not
change with L-NAME.
Chronic L-NAME:
Male and female SHR in Group 1, previously exposed to L-NAME,
gained weight during L-NAME treatment (male: 318±5 to 340±3 g; female: 205±2 to
7
212±7; p<0.05). Male SHR in Group 2, naïve to previous L-NAME, were larger than
females at baseline (315±6 vs. 185±2 g, p<0.05); neither sex gained weight during LNAME treatment (313±7 vs. 185±3 g). TTx did not alter weight gain in males (319±4 to
343±2 g; p<0.05), although female SHR receiving TTx did not gain weight (197±3 to
198±5 g).
L-NAME increases BP in male and female SHR.
Low dose L-NAME results in a greater increase in BP in female SHR.
Female and male SHR were treated with increasing doses of L-NAME and BP
was measured via telemetry (Figure 1A). BP was greater in male SHR than female
SHR at baseline (p<0.05) and the sex difference in BP was abolished upon treatment
with L-NAME. Male SHR exhibited 7%, 12% and 14% increases in BP in response to 2,
5, and 7 mg/kg/day L-NAME, respectively, compared to age-matched vehicle controls.
BP returned to vehicle control levels with termination of L-NAME treatment in males
(Figure 1B). L-NAME increased BP in female SHR 11%, 12% and 20% in response to
2, 5, and 7 mg/kg/day L-NAME, respectively, compared to vehicle controls (Figure 1C).
BP remained elevated (by 7%) 4 days after L-NAME treatment was terminated in
female SHR (Figure 1C, p=0.042). BP in vehicle control rats did not change during the
study.
Prior L-NAME exposure alters the BP response to chronic LNAME in female SHR.
BP in female SHR remained elevated 7 days after stopping the L-NAME doseresponse study; as a result there was not a sex difference in BP when starting 2 weeks
of 7 mg/kg/day L-NAME in Group 1 (Figure 2A). BP increased over the treatment
period to the same extent in both male and female SHR such that BP was comparable
8
in both sexes throughout L-NAME treatment. Male SHR in Group 2 (L-NAME naïve)
had a higher baseline BP compared to female SHR (Figure 2B).
BP significantly
increased within 24 hours of initiating L-NAME and continued to increase throughout the
treatment period, however, BP increased to a greater degree in female SHR during the
final 2 days of L-NAME treatment abolishing the sex difference in BP. The BP response
to chronic L-NAME was comparable in male SHR regardless of prior L-NAME exposure
(Figure 2C). In contrast, female SHR from Group 1 exhibited a greater increase in BP
in the first week of L-NAME treatment compared to L-NAME naïve females in Group 2
(24±3% vs. 14±2%, p<0.05; Figure 2D). BP continued to increase in both groups during
the second week of L-NAME treatment, however, the increase was greater in L-NAME
naïve female SHR, particularly in the last 2 days of treatment, (43±1% vs. 35±5%,
p<0.05) such that BPs were comparable at the end of the 2 weeks.
Chronic L-NAME induces renal injury in male and female SHR. Histological
examination of kidneys revealed that L-NAME treatment was associated with
thickening, necrosis and thrombosis of the glomerular and interstitial arteries, tubular
cast formation and fibrosis, and glomerular necrosis and ischemia in both sexes.
Kidneys of vehicle control male and female SHR were structurally unremarkable. For all
histological indices of renal injury, evidence of damage was moderate to severe in the
males treated with L-NAME and mild to moderate in L-NAME-treated females (Table 1;
Figure 3).
Urinary protein, albumin and nephrin excretion were also measured before and
after L-NAME treatment. L-NAME significantly increased urinary protein, albumin and
nephrin excretion in both sexes (Table 2). Male SHR had greater protein (p<0.01) and
9
albumin (p=0.05) excretion than females at baseline and protein excretion remained
higher in males following L-NAME treatment (p=0.07). There were no significant
differences in nephrin excretion between control or L-NAME-treated male and female
SHR or in albumin excretion following L-NAME between the sexes.
Chronic L-NAME treatment: Triple Therapy
NOS inhibition or deficiency is associated with increases in renal immune cell
infiltration (17, 18, 26). To determine the relative contribution of NOS inhibition vs. BP
elevation on renal T cell profiles in male and female SHR, additional SHR received triple
therapy (TTx) in conjunction with L-NAME to block increases in BP. TTx abolished LNAME hypertension in male and female SHR; average mean arterial pressure over the
treatment period was 131±2 mmHg in male SHR and 126±2 mmHg in female SHR.
TTx blocked L-NAME-induced increases in protein excretion in male and female SHR
and attenuated L-NAME-induced increases in albuminuria and nephrinuria.
Urinary
protein and albumin excretion were significantly less in female SHR than in males
following L-NAME + TTx (p<0.01), albuminuria was comparable between the sexes.
Chronic L-NAME treatment: T cell profile
Blood: L-NAME increased circulating CD3+ and CD4+ cells in male and female
SHR relative to vehicle-treated controls with no changes in Tregs or Th17 cells (Table
3). TTx blunted L-NAME-induced increases in circulating CD3+ and CD4+ cells and
decreased Th17 cells below vehicle control in both sexes (p<0.05).
Although not
altered by L-NAME alone TTx increased circulating Tregs relative to both L-NAME and
vehicle controls in male and female SHR (p<0.05). There were no sex differences in
10
CD3+ or Th17 absolute cell counts in whole blood, although females had more
circulating CD4+ cells and fewer Tregs under all treatments (Table 4).
Kidney: L-NAME increased renal CD3+ and Th17 cells in kidneys from male and
female SHR compared to vehicle control (p<0.001) and TTx mitigated this increase,
however, CD3+ and Th17 cells in TTx groups remained significantly greater than in
same sex vehicle controls (Figures 4A/D and 5A/D; p<0.05). TTx attenuated L-NAMEinduced increases in renal CD4+ cells in male SHR and blocked the increase in females
(Figures 4B and 5B; p<0.001).
L-NAME decreased Tregs in kidneys of male and
female SHR compared to same-sex vehicle controls (Figures 4C and 5C; p<0.05). In
male SHR, TTx increased Tregs compared to same sex vehicle and L-NAME-treated
rats (p<0.001) but in female SHR TTx only increased Tregs in comparison to same sex
vehicle rats (p<0.001). Although the overall impact of L-NAME and TTx was similar on
renal T cell profiles in male and female SHR, the magnitude of the changes differed. LNAME resulted in greater increases in CD3+ and Th17 cells in female SHR (% increase:
226% and 596%) than in males (124% and 220%) compared to same-sex vehicle
controls.
Female SHR also had a greater decrease in Tregs following L-NAME
treatment than males (% decrease: 86% vs. 49%). As a result, after L-NAME treatment
female SHR had more renal CD3+ (p=0.0009), CD4+ (p=0.0006), and Th17 cells
(p=0.0009) than male SHR. These results are supported by an analysis of absolute
numbers of renal T cells (Table 5).
Chronic L-NAME treatment: Adhesion Molecules
NO inhibits adhesion molecule expression in vitro and in vivo and decreases
vascular monocyte adhesion (9, 15).
Additional experiments assessed intercellular
11
adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) mRNA
expression in the renal cortex and medulla of male and female SHR.
L-NAME
increased renal cortical ICAM and VCAM mRNA expression in male and female SHR
and TTx abolished L-NAME induced increases in adhesion molecule expression (Figure
6). Female SHR displayed greater increases in cortical ICAM and VCAM expression
(109% and 66%) than male SHR (39% and 34%) in response to L-NAME. Medullary
ICAM and VCAM were significantly increased only in male SHR with L-NAME (240%
and 127%; Figure 7). TTx did not alter medullary ICAM expression, but abolished LNAME-induced increases in VCAM.
Discussion
The primary novel findings of the current study were that BP in female SHR is
more sensitive to NOS inhibition than in males and NOS inhibition sensitizes BP in
female SHR, but not males, to a second exposure to L-NAME. Moreover, the loss of
the sex difference in BP in male and female SHR with NOS inhibition was associated
with greater increases in renal pro-inflammatory T cells in female SHR than in males.
Finally, L-NAME-induced increases in renal T cells were dependent on both increases
in BP and NOS inhibition.
Consistent with experimental and epidemiological studies indicating greater NO
bioavailability in females relative to males (11, 14, 23), female SHR in the current study
were more sensitive to L-NAME-induced alterations in BP than males.
This was
illustrated by a number of results: female SHR exhibited a greater increase in BP to lowdose L-NAME than males thereby abolishing the sex difference in basal BP, BP in
12
female SHR did not return to basal values following the discontinuation of L-NAME, and
prior exposure to L-NAME sensitized female SHR to increases in BP to chronic NOS
inhibition. SHR are very sensitive to NOS inhibition-induced increases in BP. Although
BP plateaued at each dose in the dose-response period, we cannot rule out that
continuing treatment of lower doses of L-NAME longer than 4 days may have resulted in
additional increases in BP.
However, for the chronic phase of the study we were
interested in using a dose closer to those more commonly used in the literature to
induce L-NAME hypertension in SHR (5, 24). There is little consensus in the literature
regarding the impact of sex on BP responses to NOS inhibition. In contrast to our
findings in SHR, male Sprague-Dawley and Wistar Kyoto rats exhibited larger increases
in BP to L-NAME and Nω-nitro-L-arginine (L-NNA) than females (19, 30, 35). However,
these studies were all of longer duration (up to 24 weeks) than the current study.
Consistent with our study, female Wistar are more sensitive to L-NAME-induced
increases in BP than males over a 10 week treatment period and BP in females
remained elevated to a greater extent than in males following the termination of LNAME treatment (36).
However, two weeks of L-NAME treatment has also been
reported to result in comparable increases in BP in male and female Wistar rats (38).
Inconsistencies in the literature are likely related to the dose of NOS inhibitor used,
duration of treatment, age and strain of the animals and the method used to measure
BP.
Our study is the first to examine the contribution of NOS to BP control in
hypertensive males and females using telemetry.
Our data suggest that once the NOS system is compromised in female SHR they
lose the ability to mitigate BP increases and exhibit a BP response to NOS inhibition
13
comparable to males. Not only did BP remain elevated only in female SHR following
the termination of the L-NAME dose response experiments in Group 1, but this sexspecific carryover effect on BP reactivity in females extended for more than 10 days into
the secondary L-NAME infusion. There is indirect evidence supporting a sex difference
in the contribution of NOS to BP control in SHR. Maternal supplementation with the
NOS substrate citrulline from day 7 of gestation until 6 weeks of age resulted in a
sustained decrease in systolic BP in female SHR, although BP in males was
comparable to untreated controls by 24 weeks of age (21). One limitation of the current
study is that it is not known if sex differentially impacts the metabolism, efficacy, or
intestinal absorption of L-NAME. As such, we cannot rule out the possibility that at the
same dose of L-NAME female SHR have a greater degree of global NOS inhibition
relative to males.
Greater BP sensitivity to NOS inhibition in female SHR was paralleled by greater
increases in protein and albumin excretion, however, the degree of structural damage
following chronic L-NAME was greater in male SHR. TTx normalized L-NAME-induced
increases in protein and nephrin excretion in female SHR, suggesting that the greater
increase in protein and nephrin excretion in females results from greater increase in BP.
The acute rise in BP in females may result in a larger insult on the functional integrity of
the glomerulus that is not yet evident histologically. Greater structural alterations in
kidneys of male SHR likely reflects the fact that kidneys of male SHR are exposed to a
greater pressure load for a longer period of time compared to kidneys of females.
Contrary to our results, male Wistar rats display a greater increase in proteinuria
following chronic L-NAME (30) and male Sprague-Dawley rats are more sensitive to L-
14
NNA-induced proteinuria compared to females (35). However, in both of these studies,
the male rats also have a larger increase in BP in response to chronic NOS inhibition
which would be consistent with greater renal injury.
L-NAME hypertension increases vascular and renal monocyte/macrophage
infiltration in male SHR and contributes to end-organ damage (17, 18), yet little is known
regarding the impact of L-NAME hypertension on T cells. In the current study, chronic
L-NAME significantly increased pro-inflammatory T cells in the kidneys of SHR,
although the increase was greater in females. It should be noted that female SHR have
smaller kidneys than males and a smaller number of total cells were counted for the
flow cytometric analysis, therefore, our results may underestimate the absolute
magnitude of the sex difference in the pro-inflammatory immune cell profile following LNAME. Although not directly tested, we hypothesize that the greater increaser in proinflammatory T cells in female SHR is related to the greater BP sensitivity and increases
in renal injury. This is consistent with the finding that the lymphocyte suppressant MMF
attenuates L-NAME hypertension and renal damage in male Sprague-Dawley rats (26).
However, co-administration of MMF with L-NAME in male Munich-Wistar did not alter LNAME-induced hypertension but abolished L-NAME-induced increases in T cell
infiltration and attenuated renal injury (12), suggesting that L-NAME induced increases
in BP are independent of T cells, although renal injury is not.
To differentiate the relative contribution of increases in BP and loss of NO on
modulating T cells, additional rats were treated with TTx which implicated both a BPdependent and a NOS-dependent component to the T cell profile in SHR with L-NAME
hypertension. Interestingly, L-NAME induced increases in CD3+ and CD4+ T cells in
15
male SHR were primarily NO-dependent, while increases in CD4+ cells in female SHR
were BP dependent, suggesting sex-specific mechanisms regulate renal T cells. The
finding that TTx abolished L-NAME-induced increases in BP despite minimal decreases
in total CD3+ cells and CD4+ renal T cells calls into question the role of T cells in LNAME hypertension. However, L-NAME resulted in a BP-dependent increase in renal
Th17 cells in both sexes and Th17 cells have been directly implicated in increases in BP
(22). Therefore, measuring total CD3+ or CD4+ T cells alone may be insufficient to fully
understanding the impact of T cells on BP control. Indeed, TTx also increased renal
Tregs which limit increases in BP in male rats (2). Therefore, greater increases in Tregs
may mitigate Th17 cell-induced increases in BP.
Renal T cell profiles were only
assessed after 14 days of L-NAME treatment in the current study and L-NAME results
in a rapid rise in BP in SHR which is likely independent of the renal T-cell responses.
However, increases renal T cells could have taken place at a much earlier coinciding
with initial increases in BP, consistent with both BP and NO independently influencing
the renal T-cell profile. A limitation of the current study is that TTx itself may impact the
T cell profile. Hydralazine has been shown to reduce the number of adherent and
migrating leukocytes in the vasculature of male SHR independent on an effect on BP
(29).
To begin to assess a mechanism by which NO may impact T cell infiltration,
adhesion molecule expression was examined. Endothelial cells modulate inflammatory
cell adhesion and migration via adhesion molecules including VCAM and ICAM (6) and
NO inhibits adhesion molecule expression (9, 15). ICAM and VCAM contribute to T cell
adhesion and infiltration (25) in male rats with acute renal allograft rejection (28) and in
16
mice with lupus nephritis (39). Consistent with greater increase in T cell infiltration,
female SHR exhibited greater increases in renal cortical adhesion molecule expression
than males in the current study. Female SHR also possessed lower basal levels of
renal adhesion molecule expression which may contribute to sex difference in the T cell
profile in control rats. In contrast, only male SHR exhibited increases in medullary
VCAM or ICAM, suggesting that there are sex differences in the impact of TTx on
immune cells.
In both sexes TTx blocked L-NAME-induced increases in cortical
adhesion molecule expression, BP and protein excretion, but did not totally block T cell
infiltration.
These data suggest that BP drives the increase in VCAM and ICAM
expression and that additional adhesion factors account for the remaining increases in
CD3+ and Th17 cells in rats treated with TTx and for the robust increase in Tregs in
male SHR. Alternatively, the current study did not assess the portion of the renal T cell
profile that was due to recruitment of T cells into the kidney vs. differentiation of resident
naïve T cells.
Perspectives
There is a growing body of literature examining the role of T cells as a
determining factor in the development and progression of numerous cardiovascular
disorders in males. We recently published that immune suppression decreases BP in
female SHR (34), yet there remains a scarcity of data on the role of T cells in
hypertension in females or on how T cells are regulated between the sexes. Although
women are more likely than men to develop innate immunological disorders, including
rheumatoid arthritis and systemic lupus erythematosus (37) both of which have an
17
increased risk of cardiovascular disease (8), little is known regarding the adaptive
immune responses in women with hypertension. We propose that sex differences in the
immune cell profiles underlie sex differences in the development and progression of
many cardiovascular diseases including hypertension. Moreover, greater NO levels in
females may offer cardio-protection by suppressing the activation and infiltration of
effector T cells thereby limiting end-organ damage.
Acknowledgement
The authors would like to acknowledge the assistance of Ms. Vanessa Kemp and Ms.
Margaret Zimmerman.
Sources of Funding
The authors acknowledge funding from the American Heart Association and the
National Institutes of Health (AAE: American Heart Association SDG; KNB: American
Heart Association pre-doctoral fellowship; JCS: 1R01 HL093271-01A1 and SDG).
Conflict(s) of Interest/Disclosure(s) Statement
There are no conflicts of interest to disclose.
18
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Figure Legends
Figure 1. BP measured by telemetry in response to increasing doses of L-NAME or
vehicle in age-matched male and female SHR.
Female SHR have greater BP
sensitivity to L-NAME compared to male SHR (Panel A), although both male (Panel B)
and female SHR (Panel C) exhibit a dose-dependent increase in BP compared to same-
24
sex vehicle controls.
* indicates a significant difference from male SHR; # indicates
significant difference from same sex baseline BP and vehicle control.
Figure 2. BP in response to chronic L-NAME treatment (7 mg/kg/day) in male and
female SHR.
Chronic L-NAME significantly increased BP in both males and females
that have (Panel A) and have not (Panel B) been previously exposed to L-NAME. Prior
exposure to L-NAME did not impact BP responses to L-NAME in male SHR (Panel C).
In contrast, previous L-NAME treatment sensitized BP responses in female SHR (Panel
D). # indicates a significant difference from the baseline BP in both groups; * indicates
a significant difference from male SHR; & indicates a significant difference from agematched rat.
Figure 3. Analysis of PASH (Panels A-D) and Masson’s trichrome staining (Panels EH) in the renal cortex of male and female SHR following vehicle or L-NAME treatment at
7 mg/kg/day for 2 weeks. L-NAME induced renal injury in both male and female SHR,
however, the degree of injury was more severe in male SHR, 20x magnification; n=5.
Figure 4. Renal T cell profiles in control, L-NAME, and L-NAME + TTx treated male
SHR after 14 days of 7 mg/kg/day L-NAME. CD3+ (Panel A) and CD4+ cells (Panel B)
are expressed as % of total kidney cells, Foxp3+ (Tregs; Panel C) and ROR-γ+ (Th17;
Panel D) cells are expressed as % of CD3+CD4+ cells. L-NAME increased renal proinflammatory cells and decreased Tregs in male SHR; TTx mitigated these changes. #
indicates difference from same sex vehicle-control; † indicates significant difference
from L-NAME.
25
Figure 5. Renal T cell profiles in control, L-NAME, and L-NAME + TTx treated female
SHR after 14 days of 7 mg/kg/day L-NAME. CD3+ (Panel A) and CD4+ cells (Panel B)
are expressed as % of total kidney cells, Foxp3+ (Tregs; Panel C) and ROR-γ+ (Th17;
Panel D) cells are expressed as % of CD3+CD4+ cells. L-NAME increased renal proinflammatory cells and decreased Tregs in female SHR; TTx mitigated these changes.
# indicates difference from same sex vehicle-control; † indicates significant difference
from L-NAME.
Figure 6. ICAM (Panels A and B) and VCAM (Panels C and D) mRNA expression in
the renal cortex of male and female SHR. L-NAME-induced increases in adhesion
molecule expression in male and female SHR is blocked by TTx. # indicates difference
from same sex vehicle-control; † indicates significant difference from L-NAME.
Figure 7. ICAM (Panels A and B) and VCAM (Panels C and D) mRNA expression in
the renal medulla of male and female SHR. L-NAME increases medullary adhesion
molecule expression in male, but not female SHR.
TTx blocks L-NAME-induced
increase in VCAM, but not ICAM. # indicates difference from same sex vehicle-control;
† indicates significant difference from L-NAME.
26
Table 1.
Male
Male +
Female
L-NAME
Interstitial
Artery
Glomerulus
Female +
L-NAME
Thickening
0
2.4±0.2+
0
1.3±0.3* +
Necrosis
0
2.4±0.2+
0
1.0±0.1*+
Thrombosis
0
2.6±0.2+
0
1.0±0.1*+
Tuft necrosis
0
1.4±0.2+
0
0
Hyalinosis
0
1.4±0.2+
0
0.3±0.3*
Thrombosis
0
1.4±0.2+
0
0.3±0.3*
Renal injury scores assessed by an independent, blinded observer. Score: 0, none; 1,
mild; 2, moderate; 3, severe. * indicates significant difference from males, p<0.05; +
indicates significant difference from same sex control. N=5.
27
Table 2– Urinary excretion data to assess renal injury.
Male SHR
Female SHR
Protein
Albumin
Nephrin
excretion
excretion
excretion
(mg/day)
(mg/day)
(mg/day)
Control
20±2
0.4±0.1
0.05±0.01
L-NAME
116±23#
2.2±0.6#
0.35±0.10#
L-NAME + TTx
18±2†
1.1±0.1†
0.15±0.01†
Control
3±1
0.3±0.03
0.04±0.01
L-NAME
66±4#
2.8±0.7#
0.30±0.04#
L-NAME + TTx
3±1†
0.5±0.1†
0.17±0.02†
# indicates difference from same sex vehicle-control; † indicates significant difference
from L-NAME.
28
Table 3 – Flow Cytometry Analysis of Whole Blood.
Male SHR
Female SHR
CD3+
CD4+
Foxp3+
ROR-γ+
Control
68±1
38±2
5±1
25±1
L-NAME
77±1 #
52±2 #
4±1
28±1
L-NAME + TTx
72±1 #†
41±1†
11±1 #†
22±1†
Control
72±1
51±2
4±1
30±0.7
L-NAME
83±1 #
56±1 #
2±1
34±2
L-NAME + TTx
73±1 †
48±1 †
10±1 #†
21±2 #†
# indicates difference from same sex vehicle-control; † indicates significant difference
from L-NAME. CD3+ cells are expressed as % of total blood cells. CD4+ cells are
expressed as % of CD3+ cells. Foxp3+ (Tregs) and ROR-γ+ cells are expressed as %
CD3+CD4+ cells.
29
Table 4 – Absolute cell counts of flow cytometry analysis in whole blood.
+
All numbers * 103
Control
Male SHR
L-NAME
L-NAME +
TTx
Control
Female
SHR
L-NAME
L-NAME +
TTx
+
+
+
CD3
CD4
Foxp3
ROR-γ
68 ± 1
77 ± 1 #
72 ± 1 †
25 ± 1
40 ± 1 #
29 ± 1 †
1.3 ± 0.1
1.7 ± 0.1
3.2 ± 0.3 #†
6.5 ± 0.3
11.2 ± 0.5
6.5 ± 0.3 †
72 ± 1
83 ± 2 #
73 ± 1†
37 ± 1
46 ± 1 #
35 ± 1 †
1.5 ± 0.2
1.0 ± 0.1 #
3.6 ± 0.3 #†
10.8 ± 0.2
15.7 ± 0.8
7.4 ± 0.6 #†
All cell counts in whole blood are from an analysis of 100,000 cells in total. # indicates
difference from same sex vehicle-control; † indicates significant difference from LNAME treated rats.
30
Table 5 – Absolute cell counts of flow cytometry analysis in kidneys.
+
All numbers * 105
Control
Male SHR
L-NAME
L-NAME +
TTx
Control
Female
SHR
L-NAME
L-NAME +
TTx
+
+
+
CD3
CD4
Foxp3
ROR-γ
69 ± 6
154 ± 7 #
123 ± 9 #†
28 ± 1
79 ± 1 #
49 ± 1 †
1.3 ± 0.1
1.8 ± 0.3 #
4.3 ± 0.5 #†
1.8 ± 0.2
15.6 ± 1.0 #
4.6 ± 0.2 #†
53 ± 4
173 ± 3 #
109 ± 8 #†
24 ± 2
102 ± 2 #
54 ± 2 †
2.2 ± 0.1
1.3 ± 0.3 #
5.7 ± 0.8 #†
1.0 ± 0.1
29.8 ± 1.1 #
3.7 ± 0.5 #†
For the kidney, in males we counted 82×106 ± 5×106 total kidney cells while in females
we counted 70×106 ± 5×106 total kidney cells. # indicates difference from same sex
vehicle-control; † indicates significant difference from L-NAME treated rats.
31
Figure 1.
12 hr MAP (mmHg)
A.
180
Dose in mg/kg/day:
2
5
7
Male + L-NAME
Female + L-NAME
160
140
120
*
* * * * **L-NAME
-5
0
5
10
15
Days
12 hr MAP (mmHg)
B.
180
Dose in mg/kg/day:
2
5
160
#
7
# # #
# ##
Control Male
Male + L-NAME
## #
#
#
#
140
L-NAME
120
-5
0
5
10
15
Days
12 hr MAP (mmHg)
C.
180
Dose in mg/kg/day:
2
5
7
#
#
160
# #
#
### #
# #
Female + L-NAME
Control Female
#
#
#
#
##
140
L-NAME
120
-5
0
5
10
15
Days
1
Figure 2.
B. Group 2
220
200
180
#
#
# #
# #
# #
#
# #
# # #
Male + L-NAME
Female + L-NAME
160
140
120
-4 -2
L-NAME
0
2
4
6
8
12 hr MAP (mmHg)
12 hr MAP (mmHg)
A. Group 1
10 12 14
220
# #
200
#
#
180
#
160
140
*
**
120
-4 -2
**
***
* ** *
**
#
Naive Male + L-NAME
Naive Female + L-NAME
**
L-NAME
0
2
4
6
8
10 12 14
Days
D. Females
220
200
#
180
# #
# #
# #
#
# #
# # #
Naive Male + L-NAME
&
#
160
& &
140
L-NAME
2
4
220
200
180
#
6
8
10 12 14
#
#
# #
# # #
#
&
& & &
&
140
# #
# # #
Female + L-NAME
Naive Female + L-NAME
&
160
&
&
&
L-NAME
120
-4 -2
120
0
Male + L-NAME
12 hr MAP (mmHg)
C. Males
12 hr MAP (mmHg)
#
#
#
Days
-4 -2
# #
##
#
0
2
4
6
8
10 12 14
Days
Days
2
Figure 3.
3
Figure 4. Male SHR
B.
30
CD4+ cells
(% of total kidney cells)
CD3+ cells
(% of total kidney cells)
A.A.
#
20
# †
10
0
Control
LNAME
25
20
# †
10
LNAME + TTx
5
0
Control
LNAME
LNAME + TTx
15
# †
10
5
#
0
Control
L-NAME L-NAME + TTx
ROR- + cells
(% of CD3+ CD4+ cells)
D.
Foxp3+ cells
(% of CD3+ CD4+ cells)
C.
#
15
40
30
#
20
#†
10
0
Control
L-NAME L-NAME + TTx
4
B.
30
#
20
# †
10
0
Control
C.
Foxp3+ cells
(% of CD3+ CD4+ cells)
25
#
20
15
10
†
5
0
Control
L-NAME L-NAME + TTx
L-NAME L-NAME + TTx
D.
15
†
10
5
#
0
Control
L-NAME L-NAME + TTx
ROR- + cells
(% of CD3+ CD4+ cells)
CD3+ cells
(% of total kidney cells)
A.
CD4+ cells
(% of total kidney cells)
Figure 5. Female SHR
40
#
30
20
#†
10
0
Control
L-NAME L-NAME + TTx
5
Figure 6. Cortex
A. Male SHR
B. Male SHR
2.0
2.0
#
1.5
†
1.0
0.5
VCAM expression
(Ct)
ICAM expression
(Ct)
2.5
0.0
†
1.0
0.5
0.0
Control
LNAME
LNAME + TTx
C. Female SHR
2.5
Control
LNAME
LNAME + TTx
D. Female SHR
2.0
#
2.0
1.5
†
1.0
0.5
0.0
VCAM expression
(Ct)
ICAM expression
(Ct)
#
1.5
#
1.5
†
1.0
0.5
0.0
Control
LNAME
LNAME + TTx
Control
LNAME
LNAME + TTx
6
Figure 7. Medulla
A. Male SHR
#
3
#
VCAM expression
(Ct)
4
ICAM expression
(Ct)
B. Male SHR
3
2
1
2
†
1
0
0
Control
LNAME
Control
LNAME + TTx
C. Female SHR
LNAME
LNAME + TTx
D. Female SHR
4
2.5
VCAM expression
(Ct)
ICAM expression
( Ct)
#
3
2
1
0
2.0
1.5
1.0
0.5
0.0
Control
LNAME
LNAME + TTx
Control
LNAME
LNAME + TTx
7