Renal T cell infiltration occurs despite attenuation of development of hypertension with hydralazine in Envigo’s female Dahl rat maintained on a low-Na⁺ diet

Abstract

Many studies have suggested that renal T cell infiltration contributes to the pathogenesis of salt-sensitive hypertension. To investigate this mechanism further, we determined T cell profiles in the kidney and lymphoid tissues as a function of blood pressure in the female Envigo Dahl salt-sensitive (SS) rat maintained on low-Na⁺ (LS) diet. Mean arterial pressure and heart rate were measured by telemetry in SS rats from 1 mo old (juvenile) to 4 mo old. Normotensive salt-resistant (SR) rats were included as controls. Frequencies of T helper (CD4⁺) cells were greater in the kidney, lymph nodes, and spleen in 4-mo-old hypertensive SS rats compared with normotensive SR animals and SS juvenile rats, suggesting that renal T cell infiltration contributes to hypertension in the SS rat on a LS diet. At 1.5 mo, half of the SS rats were treated with vehicle (Veh), and the rest received hydralazine (HDZ; 25 mg·kg⁻¹·day⁻¹) for 11 wk. HDZ impeded the development of hypertension compared with Veh-treated control rats [mean arterial pressure: 157 ± 4 mmHg in the Veh-treated group (n = 6) vs. 133 ± 3 mmHg in the HDZ-treated group (n = 7), P < 0.001] without impacting T helper cell frequencies in the tissues, suggesting that HDZ can overcome mechanisms of hypertension driven by renal T cell infiltration under the LS diet. Renal frequencies of CD4⁺CD25⁺ and CD4⁺CD25⁺FoxP3⁺ regulatory T cells were significantly higher in 4-mo-old hypertensive rats compared with normotensive SR rats and SS juvenile rats, suggesting that these T cell subpopulations play a compensatory role in the development of hypertension. Greater understanding of these T cell populations could lead to new therapeutic targets for treating inflammatory diseases associated with hypertension.

INTRODUCTION

Previous study have suggest that salt-sensitive (SS) hypertension is associated with immune cell infiltration into the kidney and other target tissues. Strong evidence for this association is found in Rapp SS rats bred in Milwaukee (SS/Mcwi). A frame shift mutation in recombination activating gene 1 (Rag1^−/−) of SS/Mcwi rats resulted in significant reductions in circulating T and B cells (30). These rats were resistant to high dietary Na⁺ (4% NaCl)-induced increases in mean arterial pressure (MAP). Furthermore, SS/Mcwi rats in which the CD3-ζ chain was deleted (CD247^−/−) had a 99% reduction in circulating T cells and were also resistant to increases in MAP induced by high dietary Na⁺ (41).

After 3 wk on a high-Na⁺ diet, not only were SS/Mcwi rats hypertensive, but they also exhibited renal T cell infiltration and extensive renal pathology (30, 41). In contrast, there was less glomerular and tubular damage in SS/Mcwi rats deficient in mature or functional T cells because of Rag1^−/− (30) or CD247^−/− (41) deletion, respectively. Both of these T cell-deficient SS/Mcwi strains also had reduced urinary albumin and protein excretion rates. Pharmacological agents that inhibit immune cell infiltration into the kidney support these genetic mutation findings. Mycophenolate mofetil, which suppresses lymphocyte proliferation, reduced the magnitude of hypertension observed in the SS/Mcwi rat (29). These studies have led to the conclusion that renal infiltration of T cells is required for the full development of hypertension and renal pathology in the SS/Mcwi rat.

The Dahl SS rat was established in 1962 by Lewis Dahl after selectively breeding Sprague-Dawley rats on a high-Na⁺ diet (9, 18). In 1985, John Rapp, Jr., developed the inbred SS rat (39), which was distributed commercially by Harlan as SS/JrHsd. Although many studies have focused on mechanisms of salt sensitivity in the SS rat (35), Rapp and Dene (39) showed that both male and female SS rats also develop hypertension on a low-Na⁺ (LS) diet. When weaned at 30 days of age and thereafter maintained on a LS diet for 4 wk, systolic blood pressure in female rats was 146 mmHg, whereas on a high-Na⁺ diet, systolic blood pressure reached 209 mmHg. Previous studies of the SS rat on a LS diet have shown that the albuminuria is polygenic and that the majority of quantitative trait loci regulating albuminuria do not colocalize with quantitative trait loci regulating blood pressure (14). We have also studied SS rats on a LS diet and have shown that blood pressure increases with age more rapidly after ovariectomy and that 17β-estradiol replacement prevents this effect (15).

Phenotypic variation because of genetic differences between SS strains (2) explains why the age at which the SS rat develops hypertension on a LS diet has varied historically and by where the animals were bred (53). In 2004, we showed MAP of female SS/JrHsd rats maintained on a LS diet was around 120 mmHg at 4 mo of age (15). Last year, we reported MAP of female Envigo SS (SS/Env) rats reached nearly 150 mmHg at 4 mo of age while rats were maintained on a LS diet since weaning (34). Thus, blood pressure on a LS diet in recent female SS/Env rats is similar to the levels observed by Rapp and Dene in 1985 (39).

We have previously shown that 4-mo-old female hypertensive SS/Env rats on a LS diet had greater renal frequencies of CD4⁺ T helper (Th) cells than 1-mo-old normotensive juvenile rats (33). In this article, we investigated the role of T cells and renal infiltration in the development of hypertension on a LS diet by determining T cell profiles in the kidney and lymphoid tissues as a function of blood pressure. Antihypertensive treatment studies in most rat models of hypertension have often combined a vasodilator with a diuretic and have reported associations of T cell levels with blood pressure in treated versus control animals (1, 5, 45, 47). Here, we investigated the effect of preventing the development of hypertension on T cell frequencies by treating normotensive SS juvenile rats with the vasodilator hydralazine (HDZ). We hypothesized that impeding the development of hypertension would attenuate T cell infiltration in the kidney and result in lower frequencies of CD4⁺ T cells and Th17 cells in lymphoid tissues.

METHODS

Animals.

Female SS/Env (SS) and salt-resistant [SR/Env (SR)] inbred rats were purchased at 3–4 wk of age between November 2015 and September 2017 from Envigo (Indianapolis, IN). After weaning, animals were maintained on a LS diet (0.1% Na⁺, catalog no. 7034, Teklad; Madison, WI) while at Envigo and on a purified phytoestrogen-free LS diet (0.128% NaCl, catalog no. 120597, Teklad) (15) once at Georgetown University. Food and water was provided ad libitum. Body weight was measured weekly in all rats. Food and water intake were also measured weekly in rats that were instrumented with radio transmitters. All methods were approved by the Georgetown University Animal Care and Use Committee.

MAP and heart rate.

Four-week-old SS rats (n = 13) were anesthetized with 1–4% isoflurane at 1 l/min oxygen (Isoflurane, USP, Piramal Healthcare, Medak, Andhra Pradesh, India) and implanted with radio transmitters (catalog no. PA-C10, Data Sciences, St. Paul, MN) in a modified version of a previously described method for measuring MAP and heart rate (HR) in young rats (12). The catheter was implanted in the left femoral artery, and the battery pack was placed subcutaneously in the left flank. The analgesic carpofen (5 mg/kg, Rimadyl, catalog no. 10000319, Zoetis, Parsippany, NJ) was administered subcutaneously for up to 2 days after surgery. After recovery from surgery (7 days), MAP and HR recordings were taken every 5 min for 10 s and presented as 12-h averages using a Data Acquisition and Analysis System (Dataquest ART v4.36, Data Sciences). MAP and HR were measured at weekly intervals from 5 to 18 wk of age.

HDZ treatment.

Seven-week-old SS rats were randomized to receive either drinking water as vehicle (Veh) or HDZ (catalog no. H1753, Sigma-Aldrich; St. Louis, MO) dissolved in the drinking water; age-matched SR rats also received drinking water as Veh. The concentration of HDZ was titrated as needed to maintain the dose at 25 mg·kg⁻¹·day⁻¹ (32), which was calculated from water intake data collected during the prior week. HDZ was prepared fresh every other day. SS rats were treated with Veh or HDZ, and SR rats were treated with Veh for 11 wk before tissues were harvested for further processing at 4 mo of age.

Tissue harvest.

Four-month-old rats were anesthetized with 1–4% isoflurane. Axillary, brachial, inguinal, and lumbar (near abdominal aorta) lymph nodes (LNs) were collected as previously described (28). Briefly, a midline incision was made to open the skin layer from the suprasternal notch to the lower abdomen, exposing the LN close to the arms (axillary and brachial) and legs (inguinal). After the skin was separated from the underlying muscle, LNs were gently isolated using forceps taking care to avoid the fat while keeping the cortex of the LN intact. Isolated LNs were placed in ice-cold autoMACS Running Buffer (RB; Miltenyi Biotec, Auburn, CA), which was used as the flow cytometry buffer. After isolation and weighing, half of the spleen was placed in ice-cold RB for flow cytometry processing. Both right and left kidneys were removed and weighed. The right kidney was harvested from 4-mo-old anesthetized rats at the time of euthanasia. After decapsulation, the kidney was weighed and cut transversely into three sections. The middle section of each kidney was fixed in HistoChoice (Amresco, Solon, OH) for 16–24 h at room temperature. Fixed renal tissue was then stored in 70% ethanol until processed for histology. The right kidney poles were used for flow cytometric analysis as detailed below. Thymus and heart tissues were also isolated and weighed.

In another set of experiments, 4- to 5-wk-old juvenile SS and SR rats were anesthetized and euthanized by cardiac puncture. Both kidneys of juvenile rats were used for flow cytometric analysis. Thymus and heart tissues were also harvested and weighed.

Isolation of kidney cells for flow cytometry.

Whole kidneys from juvenile rats or right kidney poles from adult rats were stored in ice-cold HBSS containing Ca²⁺ and Mg²⁺ (catalog no. 14025076, ThermoFisher Scientific, Waltham, MA) until further processing for flow cytometry analysis. Enzyme digestion of stored kidney sections was followed by Percoll centrifugation as previously described (27, 36). Briefly, kidney sections were minced with scissors and placed in 1 ml HBSS containing 1.6 mg/ml collagenase type I-S (catalog no. C1639, Sigma-Aldrich) for 40 min at 37°C with intermittent agitation. The digested tissue was transferred to a 70-µm cell strainer (Miltenyi Biotec) and placed over a 50-ml tube, and the plunger end of a syringe was used to press the tissue through the strainer. Kidney samples were then centrifuged at 400 g for 10 min at 4°C. Samples were resuspended in 4 ml of 40% isotonic Percoll solution (catalog no. GE17-0891-02, GE Healthcare Bio-Sciences, Pittsburgh, PA) and then gently overlaid onto 80% Percoll solution. Samples were then centrifuged at 1,500 g for 30 min at room temperature with the centrifuge brake turned off. After centrifugation, adipocytes and debris were aspirated off the top layer, and mononuclear cells were collected at the interface of both solutions. Isolated cells were washed and resuspended in ice-cold RB. The cell count was then determined using a LUNA-FL Dual Fluorescence Cell Counter (Logos Biosystems, Annandale, VA) as per the manufacturer’s recommendation.

Isolation of lymphocytes and splenocytes for flow cytometry.

The LN and spleen were processed to form single cell suspensions before flow cytometric analysis. In brief, the LN and spleen were individually minced with scissors and transferred to the top of a 70-µm cell strainer (Miltenyi Biotec) placed over a 50-ml tube. The plunger end of a syringe was used to push the tissue pieces through the filter. This process was repeated at least three or four times with intermittent washes in ice-cold RB until a homogeneous suspension of single cells was achieved. These cells were then centrifuged at 400 g for 10 min at 4°C and resuspended in ice-cold RB. Red blood cell lysis was performed on spleen single cell suspensions using Red Blood Cell Lysis Buffer (catalog no. 420301, BioLegend, San Diego, CA) as per the manufacturer’s recommendation. Subsequently, cell counts of LN and spleen single cell suspensions were then determined as described above.

Analytical flow cytometry.

Phenotypic and intracellular analyses of kidney, LN, and spleen cells were performed as previously described for mice (17). Briefly, 1 million cells from each tissue type were stained for 20 min on ice in the dark with antibodies for surface markers including CD3 (pan T cells), CD4 (Th cells), CD8 (cytotoxic T cells), CD25 (IL-2 receptor α−chain), and CD11b/c (pan antigen-presenting cells). After cells had been washed, they were fixed and permeabilized using the FoxP3 Fix/Perm Buffer Set (catalog no. 421403, BioLegend) before being incubated with antibodies for intracellular proteins including FoxP3, the master regulator transcription factor commonly used to identify regulatory T (T_reg) cells (47), and related orphan receptor-γt (RORγt), the master regulator transcription factor commonly used to identify Th17 (8, 47). All antibodies were titrated to optimize multicolor flow cytometric staining. Details of antibodies used for staining are shown in Table 1.

Table 1.

Details of antibodies used for multicolor flow cytometry immune phenotyping

Antibody	Fluorochrome	Species Reactivity	Clone	Catalog No.	Vendor
CD3	VioBlue	Rat	REA223	130-102-677	Miltenyi
CD4	APC/Cy7	Rat	W3/25	201518	Biolegend
CD8a	PE-Cy7	Rat	OX-8	25-0084-82	eBioscience
CD25	PerCP-eFluor 710	Rat	OX-39	46-0390-82	eBioscience
CD11b/c	FITC	Rat	OX-42	201805	Biolegend
FoxP3	PE	Human/mouse/rat	376209	IC8970P	R&D
RORγt	APC	Human/mouse	600380	IC6006A	R&D

Vendor details regarding antibodies used for flow cytometry are shown. RORγt, related orphan receptor-γt.

OneComp eBeads Compensation Beads (catalog no. 01-1111-42, ThermoFisher Scientific, then eBioscience) were used for positive and negative staining controls and to avoid spillover signals from fluorochromes. Dead cells were excluded using the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (catalog no. L34966, ThermoFisher Scientific). Prepared samples were run on an eight-color flow cytometer (LSRFortessa, BD Biosciences, San Jose, CA) by the Flow Cytometry and Cell Sorting Shared Resource (Georgetown University), and data were collected using FACSDIVA software (BD Biosciences). Gating and flow cytometry analysis was performed in a blinded manner (by A. V. Pai) with either FCS Express 5 Flow Cytometry software (De Novo Software, Glendale, CA) or FlowJo software (FLOWJO, Ashland, OR).

Histological analysis of kidneys.

Renal tissue from SS rats stored in 70% ethanol was paraffin embedded and stained by the Histopathology and Tissue Shared Resource (Georgetown University). Masson’s trichrome staining of transverse tissue sections (3–5 µm) was also performed by the shared resource. Slides were observed and photographed using an Olympus BX43 camera at ×4 and ×20 magnification using cellSens software (Olympus Lifesciences, Waltham, MA). A semiquantitative index was used to score glomeruli in each section (~100 per rat) from 0 (best; 0% glomerular damage) to 4 (worst; 100% glomerular damage) on the basis of severity of glomerulosclerosis, as previously described (38). A score was obtained by multiplying the degree of damage by the percentage of glomeruli affected, leading to the percentage of glomerular injury. The grading of glomerular damage was performed in a blinded manner (by C. Baylis).

Statistical analyses.

Prism 7.0 (GraphPad Software, La Jolla, CA) was used to analyze all data. Flow cytometry data were first tested for normal distribution using the Shapiro-Wilk normality test. An unpaired parametric t-test was used to analyze differences between groups. A paired t-test was used to analyze chronic MAP, HR, and body weight data. Two-way ANOVA followed by Sidak’s or Tukey’s multiple comparison analysis was used as a post hoc test to analyze the effect of age and HDZ on MAP of SS rats. All data are expressed as means ± SE unless otherwise described. The significance threshold was defined as 0.05.

RESULTS

MAP and HR.

We have previously found that juvenile SS female rats at 5 wk of age were normotensive, whereas at 4 mo they were hypertensive when maintained on a LS diet since weaning (34). To examine the time course of the development of hypertension, we instrumented juvenile rats with radio transmitters and measured MAP and HR by telemetry from 5 to 18 wk. MAP increased with age and began to exceed 135 mmHg by 12 wk (Fig. 1A). In conjunction, HR began to decrease between 11 and 12 wk (Fig. 1B). In comparison, female SR rats remain normotensive even up to 10 mo of age (34).

Fig. 1.

Effect of hydralazine (HDZ) on mean arterial pressure (MAP) and heart rate (HR). Shown are MAP (A) and HR (B) data measured by radio telemetry in female salt-sensitive (SS) rats treated with vehicle (Veh; n = 6) or HDZ (n = 7) from 5 to 18 wk of age while maintained on a low-Na⁺ diet since weaning. Data are expressed as means ± SE. There was a significant effect of age (P < 0.0001) and HDZ treatment (*P < 0.0001) on MAP and on the interaction (P < 0.0001) by two-way ANOVA. There was a significant effect of age (P < 0.0001) on HR but no effect of HDZ or on the interaction by two-way ANOVA.

HDZ in conjunction with hydrochlorothiazide and reserpine have been previously shown to attenuate the development of hypertension in SS rats (23). To determine if HDZ alone was sufficient to achieve this effect, we instrumented juvenile rats with radio transmitters and measured MAP and HR by telemetry during treatment with HDZ from 5 to 18 wk. HDZ treatment alone was able to attenuate the age-associated increase in MAP (Fig. 1A). MAP in 18-wk-old HDZ-treated SS rats was 24 mmHg less than MAP of Veh-treated rats. HDZ had no effect on the age-associated decrease in HR from 5 to 18 wk of age (Fig. 1B).

Body and tissue weights, food and water intake, and hematocrit.

There was no detectable effect of HDZ treatment on body weight. HDZ also had no effect on thymus wet weight; however, tissue wet weights of the kidney (1.2-fold), heart (1.1-fold), and spleen (1.1-fold) were slightly higher in HDZ-treated SS rats compared with Veh-treated control rats (Table 2). HDZ-treated rats drank 35% less water and ate slightly less food (−0.5 g) per day compared with Veh-treated rats (Table 2). There was no effect of HDZ on hematocrit.

Table 2.

Effects of HDZ on body and tissue weights, food and water intake, and hematocrit

Measurement	Veh-Treated SR Group	Veh-Treated SS Group	HDZ-Treated SS Group
Initial body weight, g		114 ± 2	118 ± 2
n		7	6
Final body weight, g	228 ± 5	250 ± 6†	240 ± 4
n	8	7	6
Thymus, g/100 g	0.11 ± 0.004	0.14 ± 0.01†	0.15 ± 0.01
n	8	7	6
Left kidney, g/100 g	0.31 ± 0.01	0.32 ± 0.01	0.39 ± 0.01*
n	8	7	6
Right kidney, g/100 g	0.32 ± 0.01	0.32 ± 0.01	0.39 ± 0.01*
n	8	7	6
Spleen, g/100 g	0.25 ± 0.006	0.26 ± 0.004	0.28 ± 0.01*
n	8	7	6
Heart, g/100 g	0.30 ± 0.01	0.37 ± 0.01†	0.40 ± 0.01*
n	8	7	6
Food intake, g/day		15 ± 0.3	15 ± 0.4*
n		7	6
Water intake, ml/day		20 ± 0.8	13 ± 0.7*
n		7	6
Hematocrit, %	42.7 ± 1	48.6 ± 0.6†	47.8 ± 2
n	3	5	4
Initial systolic blood pressure, mmHg		115 ± 2	115 ± 2
n		7	6
Initial diastolic blood pressure, mmHg		75 ± 6	82 ± 1
n		7	6
Initial mean arterial pressure, mmHg		99 ± 2	100 ± 1
n		7	6
Final systolic blood pressure, mmHg		186 ± 4	160 ± 4*
n		7	6
Final diastolic blood pressure, mmHg		127 ± 3	111 ± 5*
n		7	6
Final mean arterial pressure, mmHg		157 ± 4	133 ± 3*
n		7	6

Data are expressed as means ± SE; n, number of rats. Female salt-resistant (SR) rats were treated with vehicle (Veh; n = 8), and female salt-sensitive (SS) rats were treated with Veh (n = 6) or hydralazine (HDZ; n = 7) at 7 wk of age. Treatments lasted 11 wk, and all rats were maintained on a low-Na⁺ diet since weaning. Body weight was determined initially at 5 wk of age and at 18 wk of age. Wet tissue weights of the thymus, kidney, spleen, and heart were determined at 18 wk. Food and water intake were measured weekly, and the average daily consumption is reported. Hematocrit was determined at 18 wk.

^*P < 0.05 vs. Veh treatment of the SS strain by a Student's t-test;

^†P < 0.05 vs. Veh treatment of the SR strain by a Student's t-test.

Compared with SS rats, female SR rats weighed (228 ± 5 g, n = 8) ~10% less at 12 wk of age. Tissue wet weights of the thymus (0.11 ± 0.004, n = 8) and heart (0.30 ± 0.01, n = 8) were also slightly lower, whereas other tissues had comparable wet weights.

Th cell and cytotoxic T cell populations.

We have recently shown striking differences in renal Th (CD4⁺) and cytotoxic T (CD8⁺) cell populations between juvenile SR and SS female rats (33). Even though 5-wk-old SS rats were normotensive, the Th cell population was higher and cytotoxic T cell population was lower in the kidney compared with age-matched SR rats. At 4 mo, these differences in the renal T cell profile between SR and SS rats were magnified. To determine whether the age-associated increase in blood pressure in SS rats caused the changes in the T cell profile, we examined the effect of attenuating the development of hypertension on the T cell profile independently of blood pressure and independently of high Na⁺ because these experiments were conducted on a LS diet. We measured the frequencies of Th and cytotoxic T cells in target tissues by multiflow cytometry in Veh- or HDZ-treated SS rat tissues and compared these findings with age-matched normotensive SR rats.

Flow analysis was performed by first creating a lymphocyte gate based on known characteristics of lymphocyte size and granularity to eliminate other cell types from the analysis (Fig. 2A). In some analyses, the lymphocyte gate was followed by single cell selection (Fig. 2B) and exclusion of dead cells (Fig. 2C). CD3⁺ T cells were gated based on identifying live cells or directly from the lymphocyte gate (Fig. 2D) and further characterized into CD4⁺ and CD8⁺ T cells based on antibody staining (Fig. 2E). After the CD4⁺ population was isolated (Fig. 2), gates for IL-2 receptor CD25 and the transcription factor FoxP3 were set (Fig. 3A); CD25 and FoxP3 are markers used to identify activated Th (CD4⁺ CD25⁺) and T_reg (CD4⁺ FoxP3⁺ and CD4⁺ CD25⁺ FoxP3⁺) cells. The fluorescence minus one (Fmo) control for FoxP3 (Fig. 3B) was used as a guide to set gates for FoxP3⁺ staining (Fig. 3C).

Fig. 2.

Flow cytometry gating strategies for T helper and cytotoxic T cell populations. An initial lymphocyte gate was chosen based on cell size and volume (A) from which single cells were selected (B) followed by identification of live cells (C). The pan T cell population (CD3⁺) was then gated (D) and further characterized into CD4⁺ and CD8⁺ T cells based on anti-CD4⁺ and anti-CD8⁺ antibody staining (E). Data are representative of over three experiments conducted by multichannel flow cytometry.

Fig. 3.

Flow cytometry gating strategies for regulatory T cell populations. Shown are representative flow cytometry gating images of sample (A), floursence minus one (Fmo) control (B), and overlay plots (C) for CD4⁺CD25⁺FoxP3⁺ regulatory T cells. Data are representative of over three experiments conducted by multichannel flow cytometry.

Although HDZ treatment for 11 wk attenuated the development of hypertension (Fig. 1A), there was no effect on the pan T cell population in the kidney (Fig. 4A), LN (Fig. 4E), and spleen (Fig. 4I) compared with Veh-treated SS rats. Furthermore, there were no detectable effects of HDZ on the frequency of Th (Fig. 4, B, F, and J) or cytotoxic T cell populations (Fig. 4, C, G, and K) in the kidney (Fig. 4, B and C), LN (Fig. 4, F and G), and spleen (Fig. 4, J and K), nor did HDZ have any effect on the ratio of CD4 to CD8 in these tissues (Fig. 4, D, H, and L).

Fig. 4.

Effects of hydralazine (HDZ) on T helper and cytotoxic T cell populations in the kidney, lymph node, and spleen. Shown are frequencies of pan CD3⁺ (A, E, and I), CD4⁺ (B, F, and J), and CD8⁺ (C, G, and K) T cell populations and the CD4⁺-to-CD8⁺ ratio (D, H, and L) in the kidney (A–D), lymph node (E–H), and spleen (I–L) in female rats at 18 wk of age. Salt-resistant (SR) rats were treated with vehicle (Veh; n = 8), and salt-sensitive (SS) rats were treated with Veh (n = 6) or HDZ (n = 7). Treatments lasted 11 wk, and all rats were maintained on a low-Na⁺ diet since weaning. Data are expressed as means ± SE. $P < 0.05 vs. SR rats with the same treatment and *P < 0.05 vs. vehicle-treated rats of the same strain by an unpaired Student’s t-test.

Similar to our previous findings (33) in the kidneys of 4-mo-old SR and SS rats (Fig. 4, A–D), the frequency of pan CD3⁺ T cells in the LN (Fig. 4E) was comparable between normotensive Veh-treated SR rats and hypertensive SS Veh-treated rats at 18 wk; however, SS rats had a slightly lower frequency of CD3⁺ T cells in splenocytes (Fig. 4I) than Veh-treated SR control rats. In contrast, frequencies of CD4⁺ Th cells in the LN (Fig. 4F) and splenocytes (Fig. 4J) were all markedly higher in Veh-treated SS rats compared with female Veh-treated SR rats, whereas frequencies of CD8⁺ cytotoxic T cells in the LN (Fig. 4G) and splenocytes (Fig. 4K) were all significantly lower in Veh-treated SS rats compared with Veh-treated SR rats. This distinct pattern of Th and cytotoxic T cells resulted in nearly a twofold higher CD4-to-CD8 ratio in Veh-treated SS rat tissues compared with Veh-treated SR rat tissues (Fig. 4, D, H, and L).

CD4⁺RORγt⁺ Th17 cell populations.

Mice deficient in IL-17 have been shown to be protected from the development of hypertension (24). Therefore, we investigated whether or not the frequency of Th17⁺ cells (CD4⁺RORγt⁺) differed between normotensive SR and hypertensive SS rats and whether this T cell population was altered by preventing the development of hypertension in SS rats. No differences were observed in the frequency of Th17 cells in the LN and kidney of 18-wk-old hypertensive SS and normotensive SR rats [Th17 (percentage of CD3⁺CD4⁺ cells): Veh-treated SR rats 10 ± 2.1 (n = 8) vs. Veh-treated SS rats 9.2 ± 1.8 (n = 7) in the LN, P = 0.75; and Veh-treated SR rats 5.0 ± 1.9 (n = 8) vs. Veh-treated SS rats 5.1 ± 1.2 (n = 7) in the kidney, P = 0.96]. Furthermore, HDZ had no effect on the frequency of Th17 cells in the kidney [Th17 (percentage of CD3⁺CD4⁺ cells): Veh-treated SS rats 5.1 ± 1.2 (n = 7) vs. HDZ-treated SS rats 5.9 ± 2.5 (n = 6), P = 0.78] or LN [Th17 (percentage of CD3⁺CD4⁺ cells): Veh-treated SS rats 9.2 ± 1.8 (n = 7) vs. HDZ-treated SS rats 8.4 ± 3.1 (n = 6), P = 0.82]. Th17 cells in splenocytes were below the detection limit of the flow cytometry.

Activated Th and T_reg cell populations.

Activated Th (CD4⁺CD25⁺) and T_reg (CD4⁺FoxP3⁺ and CD4⁺CD25⁺FoxP3⁺) cells are specific populations of T cells that have been associated with hypertension and hypertensive heart disease in mice (4, 19, 20). Therefore, we investigated whether or not the frequency of these T cell populations differed between normotensive SR and hypertensive SS rats and whether these T cell populations were altered by preventing the development of hypertension in SS rats.

At 4 mo, Veh-treated SS rats had significantly higher frequencies of CD4⁺CD25⁺ T cells in the kidney (Fig. 5A), LN (Fig. 5D), and splenocytes (Fig. 5G) compared with Veh-treated SR rats. Moreover, the frequencies of CD4⁺Foxp3⁺ T_reg cells in LNs (Fig. 5E) and splenocytes (Fig. 5H) were markedly higher in hypertensive Veh-treated SS rats compared with normotensive Veh-treated SR rats. Although no differences in CD4⁺Foxp3⁺ T cells were observed in kidney cells (Fig. 5B) between Veh-treated SR and SS rats, the frequency of CD4⁺CD25⁺Foxp3⁺ T cells was markedly higher in Veh-treated SS rats compared with Veh-treated SR rats in the kidney (Fig. 5C), LN (Fig. 5F), and spleen (Fig. 5I).

Fig. 5.

Effects of hydralazine (HDZ) or vehicle (Veh) on activated T cells and regulatory T cell populations in the kidney, lymph node, and spleen. Shown are frequencies of CD4⁺CD25⁺ activated T cells (A, D, and G) and CD4⁺FoxP3⁺ (B, E, and H) and CD4⁺CD25⁺FoxP3⁺ (C, F, and I) regulatory T cell populations expressed as percentages of the CD4⁺ T helper cell population in the kidney (A–C), lymph node (D–F), and spleen (G–I) in female rats at 18 wk of age. Salt-resistant (SR) rats were treated with Veh (n = 8), and salt-sensitive (SS) rats were treated with Veh (n = 6) or HDZ (n = 7). Treatments lasted 11 wk, and all rats were maintained on a low-Na⁺ diet since weaning. Data are expressed as means ± SE. $P < 0.05 versus SR rats with the same treatment and *P < 0.05 versus Veh-treated rats of the same strain by an unpaired Student’s t-test.

HDZ had no effect on the frequency of activated Th cells (CD4⁺CD25⁺) in the LN (Fig. 5D) and spleen (Fig. 5G). There was also no effect of HDZ on the frequency of either CD4⁺FoxP3⁺ (Fig. 5, B, E, and H) or CD4⁺CD25⁺FoxP3⁺ (Fig. 5, C, F, and I) T_reg populations in the kidney (Fig. 5, B and C), LN (Fig. 5, E and F), and spleen (Fig. 5, H and I) compared with Veh-treated control rats, whereas the frequency of activated Th cells in the kidney was slightly diminished by HDZ treatment (Fig. 5A).

We have previously shown that age increases the difference in renal Th cell frequencies between SS and SR rats; although the frequency of the renal CD4⁺ population was 1.2-fold greater in SS versus SR rats at 1 mo, by 4 mo of age, the frequency was 1.4-fold greater (33). To see if age also amplified the difference in frequency of activated T and T_reg cells, we compared the frequencies of these T cell populations in the kidney of juvenile SS and SR rats. The renal frequency of CD4⁺CD25⁺ T cells was 1.5-fold higher in SS rats compared with SR rats at 1 mo of age, whereas the frequency of renal CD4⁺FoxP3⁺ and CD4⁺CD25⁺FoxP3⁺ T cells was 1.6- and 1.3-fold higher, respectively, in SS rats compared with SR rats (Fig. 6A). Thus, age magnified the difference in renal frequencies in CD4⁺CD25⁺ and CD4⁺CD25⁺FoxP3⁺ populations between SS and SR rats but not in CD4⁺FoxP3⁺ T_reg cells (Fig. 6B).

Fig. 6.

Effects of age on activated T cells and regulatory T cell populations in the kidney. Shown are frequencies of CD4⁺CD25⁺ activated T cells (A) and CD4⁺FoxP3⁺ (B) and CD4⁺CD25⁺FoxP3⁺ (C) regulatory T cell populations expressed as percentages of the CD4⁺ T helper cell population in 5-wk-old (n = 8 rats/group) and 18-wk-old (n = 7 rats/group) female salt-sensitive (SS) and salt-resistant (SR) rats maintained on a low-Na⁺ diet since weaning. Data are expressed as means ± SE. $P < 0.05 vs. SR rats of the same age and #P < 0.05 vs. 5 wk of age of the same strain by an unpaired Student’s t-test.

Glomerular injury.

We have previously shown that the 4-mo-old hypertensive SS rat exhibits mild glomerular injury (34). Thus, we investigated whether or not this mild glomerular injury was reduced under conditions in which the development of hypertension was attenuated by 11 wk of HDZ treatment. HDZ-treated rats exhibited slightly more overall glomerular damage (Fig. 7A) than Veh-treated control rats, although this trend did not reach statistical significance (Fig. 7B and Table 3). Furthermore, the amount of overall renal tubular damage observed was mild with only minor focal tubule-interstitial fibrosis (Fig. 7A). Thus, the amount of renal injury was minimal compared with what is observed in the kidneys of SS rats treated with a high-Na⁺ diet (10).

Fig. 7.

Effect of hydralazine (HDZ) on renal injury. A and B: representative images of renal pathology (A) and quantification of glomerular injury (B) in transverse sections of right kidneys. Images were taken at ×4 and ×20 magnification of glomeruli and interstitial tubular space from 18-wk-old female salt-sensitive (SS) rats treated with vehicle (Veh; n = 6) or HDZ (n = 7). Treatments lasted 11 wk, and all rats were maintained on a low-Na⁺ diet since weaning. Data are expressed as means ± SE. *P < 0.05 vs. Veh treatment by an unpaired Student’s t-test.

Table 3.

Effect of HDZ on histological glomerular injury

	Injury Grade					Tubular injury	Number of casts
	0	1+	2+	3+	4+
Vehicle-treated SS group	88 ± 4.6	6.7 ± 1.2	0.1 ± 0.1	0.1 ± 0.1	0.9 ± 0.3	Very mild	9.1 ± 1
HDZ-treated SS group	86 ± 6.1	4.7 ± 1.3	0.5 ± 0.5	0.5 ± 0.5	2 ± 0.4*	Very mild	6.5 ± 0.8

Data are means ± SE. Female salt-sensitive (SS) rats were treated with vehicle (n = 6) or hydralazine (HDZ; n = 7) for 11 wk while maintained on a low-Na⁺ diet since weaning. Data are based on a glomerular injury scale of 0–4+, where 0 = no damage, 1+ = focal damage involving <25% of the glomerulus, 2+ = focal damage between 25% and 50% of the glomerulus, 3+ = widespread damage involving 50–75% of the glomerulus, and 4+ = widespread damage involving 75–100% of the glomerulus and includes obsolescent glomeruli. A description of tubular injury and numbers of casts are also shown; the area of casts was not taken into consideration.

^*P < 0.05 vs. vehicle treatment of the same injury grade by an unpaired Student’s t-test.

DISCUSSION

A major finding of the present study is the positive association between the renal frequencies of Th cells and the development of hypertension in the SS rat while maintained on a LS diet. The frequency of the renal CD4⁺ T cell population was higher in the hypertensive SS rat compared with the normotensive SR rat at 4 mo of age (Fig. 4). Similar findings were observed in the LN and spleen. These data expand on our previous work in the Dahl SS/Env rat showing that 4-mo-old female rats had greater renal frequencies of CD4⁺ Th cells than 1-mo-old normotensive juvenile rats (33). These data support previous research in the SS rat bred in Milwaukee. Studies in that animal have shown the absence of functional T cells reduced the magnitude of hypertension and renal injury induced by dietary Na⁺, demonstrating that renal T cell infiltration contributes to the pathogenesis of SS hypertension (30, 41). Our study suggests renal T cell infiltration can also contribute to the pathogenesis of hypertension in the absence of a high-Na⁺ diet in the SS rat bred at Envigo. Studies in another model of hypertension, i.e., the spontaneously hypertensive rat (SHR), support this conclusion. Reducing immune cell infiltration in the SHR kidney via mycophenolate mofetil treatment was associated with lower blood pressure (48). Using whole genome sequencing, we showed that SS/Env rats (marketed as SS/JrHsd) has a higher rate of heterogeneity compared with SS/Mcwi rats bred at Milwaukee and SS/Jr rats bred at the University of Toledo (34). Thus, consomic studies of these diverse strains, including the SHR, may prove useful in ultimately identifying the genes responsible for renal Th cell infiltration and the association with hypertension.

The second major finding of the present study is that HDZ had little effect on the CD4⁺ T cell population even though this vasodilator was able to markedly attenuate the development of hypertension (Fig. 1). These findings support prior studies in the SHR. Tipton et al. (47) found that attenuating the development of hypertension in the SHR using the diuretic hydrochlorothiazide had no impact on the degree of renal infiltration of Th (CD4⁺) and cytotoxic T (CD8⁺) cells in either sex. Previous studies in the SHR have also shown that reserpine and HDZ attenuated the development of hypertension but had minimal effects on renal T cell infiltration. These observations indicate that vasodilators and diuretics are able to overcome the mechanisms by which renal T cell infiltration drives the development of hypertension. These findings may explain why vasodilators and diuretics can be effective antihypertensive agents in the presence of renal inflammation.

Renal perfusion pressure is one mechanism that could lead to renal T cell infiltration. Elegant studies in the hypertensive SS/Mcwi rat using an electronic servocontrol technique showed increased renal perfusion pressure triggered renal T cell infiltration (11). Although we cannot rule out the role of increased renal perfusion pressure in contributing to renal T cell infiltration during the development of hypertension in the Veh-treated SS/Env rat, other mechanisms are likely to play a role in the presence of HDZ because HDZ has been shown in several studies to reduce the glomerular filtration rate and increase renal blood flow in SS (21) and other animal models (6, 49). Furthermore, unlike the renal damage observed in SS rats after maintenance on a high-Na⁺ diet that might also contribute to mechanisms of T cell-mediated hypertension (30, 41), the hypertensive 4-mo-old SS rats on the LS diet had only mild evidence of glomerular and tubular damage. Furthermore, as observed in the SHR (42), HDZ had no effect on renal injury (Fig. 7).

Greater activity of the renin-angiotensin system is another possible mechanism driving renal T cell infiltration. Inhibition of ANG II actions by pharmacologically blocking the ANG II type 1 receptor (AT₁R) with an antagonist has been shown to reduce renal T cell infiltration (31, 50). Furthermore, we have shown that SS rats have higher AT₁R binding in the kidney compared with SR rats while maintained on a LS diet (52). Thus, increased renal AT₁R activity may be one mechanism contributing to the higher frequencies of renal Th cell infiltration in 4-mo-old SS versus SR rats and why these T cell subpopulation frequencies increase with age.

Some studies have suggested Th17 cells contribute to the development of hypertension. Male and female SHRs have higher renal frequencies of Th17 (CD4⁺RORγt⁺) cells than their normotensive Wistar-Kyoto counterparts (47). Furthermore, IL-17 null mice did not reach the magnitude of ANG II-induced hypertension compared with wild-type littermates (24). The Th17 population, however, is less likely to make a critical contribution to the pathogenesis of hypertension on a LS diet in SS rats because no differences were observed between the frequency of Th17 cells in the kidneys of 18-wk-old hypertensive SS and normotensive SR rats.

The third major finding of the present study is the positive association between blood pressure and renal frequencies of CD4⁺CD25⁺ activated T cell and the CD4⁺CD25⁺FoxP3⁺ T_reg populations. The frequency of these T cell subpopulations was higher in the kidney, LN, and spleen in hypertensive SS rats compared with normotensive SR rats at 4 mo of age (Fig. 5), and the renal frequencies of these populations were also higher than in normotensive SS juvenile rats (Fig. 6).

CD25 is the α-chain of the IL-2 receptor (13), and surface expression of CD25 on T cells is a sign of proliferation or activation. Some studies have suggested that CD25⁺ T cell populations have antihypertensive and antifibrotic actions; however, study results have been mixed. Studies in male C57/BL6 mice demonstrated that adoptive transfer of CD4⁺CD25⁺ T cells attenuate the magnitude of hypertension induced by either ANG II or aldosterone (4, 20). In contrast, adoptive transfer of CD4⁺CD25⁺ T cells to ANG II-infused hypertensive mice was shown to improve microvascular function (37) and cardiac damage (22) independently of high blood pressure. Studies in the SHR have offered different conclusions regarding the role of activated T cells in the development of hypertension. One group reported that male SHRs express less than half the number of CD4⁺CD25⁺ T cells in the spleen than normotensive Wistar-Kyoto rats, which they interpreted as suggesting that lack of this population contributes to the development of hypertension in the SHR (51). The protective or detrimental actions of these activated T cells may depend on whether they secrete anti-inflammatory (IL-10 and transforming growth factor-β) or proinflammatory (IL-6, interferon-γ, and TNF-α) cytokines. Further investigation into the lineage of the activated T cells would help in understanding the molecular mediators involved in this mechanism.

Many studies have suggested that FoxP3⁺ T_reg populations play an antihypertensive role and protect target tissues from injury. A specific study (19) of the CD4⁺CD25⁺FoxP3⁺ T_reg population showed that adoptive transfer of these cells into male C57/BL6 mice resulted in markedly less cardiac fibrosis induced by transaortic constriction. A novel cytokine immune complex of IL-2 and anti-IL-2 monoclonal antibody mix was used to induce CD4⁺CD25⁺Foxp3⁺ T_reg cells in C57BL/6J mice. Although the immune complex effectively induced T_reg expansion by fivefold, it had no effect on the magnitude of ANG II-induced hypertension (26). Corollary experiments designed to reduce specific populations of T_reg cells are experimentally more difficult. A recent report (7) in mice showed that anti-CD25 treatment abolished the protective effects of knocking out both complement receptors 3a and 5a in the development of ANG II-induced hypertension. However, in addition to depleting the CD25⁺FoxP3⁺ T_reg cells, this antibody treatment also reduced other cell populations that express CD25, including memory T cells, activated B cells, and subtypes of thymocytes, myeloid precursors, and oligodendrocytes that express CD25.

One laboratory reported that both male and female SHRs have higher levels of CD4⁺FoxP3⁺ T cells than their Wistar-Kyoto counterparts (47). Furthermore, there was a positive correlation between the frequencies of CD4⁺FoxP3⁺ T cells and the development of hypertension in the female SHR; the frequency of T_reg cells increased along with the rising blood pressure. This laboratory also reported sex differences in T cell profiles after treating SHRs with a combination of the diuretic hydrochlorothiazide and the vasodilator reserpine. Although 6 wk of hydrochlorothiazide treatment prevented the age-associated increase in blood pressure in both male and female SHRs, this treatment led to lower frequencies of CD4⁺FoxP3⁺ T cells in the kidney of female rats with no effect on this population in the male kidney. One interpretation of these data is that this T_reg population is a sex-specific compensatory response to the mechanisms underlying the development of hypertension in the female SHR. Our findings in the SS rat support the existence of a compensatory CD4⁺CD25⁺FoxP3⁺ T_reg response in the female rat (Figs. 5 and 6).

Studies of T_reg frequencies in the kidney, LN, and spleen have varied significantly depending on the animal model of hypertension. Although these differences could be model specific, some of these reported differences could in part be because of different gating and compensation strategies used in flow cytometry (25). Few details are provided in methods sections, so it is difficult to determine the role of gating strategies in these differences. The largest factor may be in the definition of T_reg cells. Phenotypical characterization of T_reg cells varies among investigators studying rats. Although some investigators have reported the frequency of CD4⁺CD25⁺FoxP3⁺ T_reg cells, not all T_reg cells express CD25. In fact, roughly 40% of T_reg cells do not express CD25 in the blood, LN, and spleen of Wistar rats (40).

Few immune markers have been characterized for rats compared with mice, which is mainly because of the lower availability of genetically modified rats and rat‐specific monoclonal antibodies compared with mice (40). CD25 has been frequently cited as a marker for T_reg cells; however, this is true only for mice and not humans or other animals (3, 46). High expression of this marker (CD25^high) on the cell surface rather than the presence or absence of CD25⁺ is a more accurate marker of T_reg cells (44). CD4⁺CD25^high cells are T_reg cells, while the CD4⁺CD25⁺ population more aptly reflects an activated T cell population (44). Rodriguez-Perea et al. (40) also reported that there is a strong correlation (r > 0.7) between the frequencies of CD25⁺FoxP3⁺ and CD4⁺FoxP3⁺ T cells in the LN, spleen, and circulation of rats, suggesting that these markers are more accurate identifiers of T_reg cell populations than CD4⁺CD25⁺ T cells.

In the present study, we did not observe any differences in the frequency of CD4⁺FoxP3⁺ T_reg populations in the kidney of 1-mo-old normotensive juvenile and 4-mo-old old hypertensive SS rats (Fig. 6), nor did we see any effect of HDZ on the frequency of these T_reg populations in the kidney, LN, or spleen (Fig. 5). Although the frequency of CD4⁺CD25⁺FoxP3⁺ T_reg cells was almost twofold higher in the kidney of 4-m-old hypertensive SS rats compared with 1-mo-old juvenile rats (Fig. 6), HDZ had no effect on these cells in kidney or lymphoid tissues (Fig. 5). There seemed to be two distinct ranges of CD4⁺CD25⁺FoxP3⁺ T_reg frequencies; however, no apparent trend was observed between the T cell frequencies and actual MAP values. Thus, we cannot rule out that the increased frequency of CD25⁺FoxP3⁺ Th cells could be a compensatory response to increasing blood pressure and related renal injury in the development of hypertension in the female SS/Env rat.

We have previously shown sex-specific T cell effects in a model of ANG II-induced hypertension (17). Although adoptive transfer of male T cells exacerbated the magnitude of hypertension-induced by ANG II in the male B6.Rag1^−/− mouse, equivalent numbers of female T cells did not. Both CD4⁺ and CD8⁺ T cell subsets showed this same sex-specific effect on ANG II-induced hypertension (43). Unfortunately, our recent work suggests that a genetic drift has occurred in the B6.Rag1^−/−-null mouse, resulting in a loss of resistance to ANG II-induced hypertension (16). Therefore, studying the role of biological sex in the contribution of specific T cell subtypes in the pathogenesis of ANG II-dependent hypertension in this mouse model has new limitations.

In summary, the major finding of our study is the positive association between the renal frequencies of Th cells and hypertension, suggesting that renal T cell infiltration contributes to the pathogenesis of hypertension in the SS/Env rat in the absence of high dietary Na⁺. HDZ had little effect on the CD4⁺ T cell population even though this vasodilator was able to attenuate markedly the development of hypertension, indicating this vasodilator can overcome CD4⁺-mediated mechanisms of hypertension. Finally, we found that CD4⁺CD25⁺ activated T cell and CD4⁺CD25⁺FoxP3⁺ T^reg cell populations positively correlated with blood pressure, suggesting they may play a compensatory role in the development of hypertension on a LS diet in the SS rat. It will be important to study these T cell populations further to elucidate the diverse T cell mechanisms modulating hypertension and associated end-organ damage. Greater understanding could lead to new therapeutic targets for treating this devastating and prevalent disorder.

GRANTS

This work was supported by National Institutes of Health Grants TL1-TR-001431 (to A. Pai), UL1-TR-001409 (to K. Sandberg), and R01-HL-119380 (to K. Sandberg and H. Ji).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

A.V.P. and K.S. conceived and designed research; A.V.P., C.A.W., A.M.A.d.S., P.S.K., E.J.P., D.A.W.Jr., J.L., H.J., X.W., M.J.Z., and C.B. performed experiments; A.V.P., C.A.W., D.A.W.Jr., J.L., H.J., M.J.Z., C.B., and K.S. analyzed data; A.V.P., C.A.W., A.M.A.d.S., P.S.K., H.J., C.B., and K.S. interpreted results of experiments; A.V.P. prepared figures; A.V.P., C.A.W., and K.S. drafted manuscript; A.V.P., C.A.W., A.M.A.d.S., P.S.K., H.J., C.B., and K.S. edited and revised manuscript; A.V.P., C.A.W., A.M.A.d.S., P.S.K., E.J.P., D.A.W.Jr., J.L., H.J., X.W., M.J.Z., C.B., and K.S. approved final version of manuscript.