The fluorochrome-conjugated monoclonal antibodies (mAbs) to rat antigens for flow cytometric analysis, including anti-CD45 FITC (Cat. no. 202205), anti-CD3 FITC (Cat. no. 201403), anti-CD4 APC (Cat. no. 201509), anti-CD8a PE (Cat. no. 200608), anti-CD25 PE (Cat. no. 202105), mouse FITC-IgG1, κ Isotype Ctrl (Cat. no. 400107), mouse FITC-IgM, κ Isotype Ctrl (Cat. no. 401606), mouse APC-IgG1, κ Isotype Ctrl (Cat. no. 400122), mouse PE-IgG2a, κ Isotype Ctrl (Cat. no. 400212) and mouse PE-IgG1, κ Isotype Ctrl (Cat. no. 981804) were obtained from BioLegend (San Diego, CA, USA). Anti-Cx43 antibody (Cat. no. ab79010) was obtained from Abcam (Cambridge, MA, USA). The goat anti-mouse IgG/FITC secondary antibody (Cat. no. 405305) was acquired from BioLegend, and 10X erythrocyte lysis buffer was supplied by Mindray (Shenzhen, China). Rat IL-2 and rat IL-6 enzyme-linked immunosorbent assay (ELISA) kits were provided by MultiSciences Biotech Co., Ltd. (Hangzhou, China). RPMI-1640 medium was obtained from Gibco (Grand Island, NY, USA) and fetal bovine serum (FBS) was acquired from HyClone (Logan, UT, USA). Penicillin, streptomycin, concanavalin A (ConA) and TRI reagent were obtained from Sigma-Aldrich (St. Louis, MO, USA). Gap27 was supplied by Apexbio (Boston, MA, USA), and the cell counting kit-8 (CCK-8) kit was provided by Zomanbio (Beijing, China). The PrimeScript™ RT reagent kit was acquired from Takara (Shiga, Japan), and the QuantiNova SYBR-Green PCR kit was obtained from Qiagen (Hilden, Germany). Primers were synthesized by Sangon Biotech (Shanghai, China).

Age-matched 16- to 17-week-old male spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats (n=15 in each group) were used in this study. The experimental animals were purchased from vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The protocol of this study was approved by the Institutional Animal Care and Use Committees (IACUC) of the Medical College of Shihezi University, Shihezi, China. The study was performed in accordance with the guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Public Health Service Policy on Humane Care and Use of Animals, DHEW Publication no. 96-01, PHS Policy revised in 2002).

The systolic blood pressure (SBP) was measured non-invasively using a tail-cuff apparatus (Chengdu Thai Union Co., Chengdu, China) prior to the experiment. The rats were placed for 10 min in a chamber heated to 37°C. A cuff equipped with a pneumatic pulse sensor was wrapped around the tail of the rat and was placed in an individual plastic restrainer. Blood pressure was accurately recorded for each rat and averaged from at least 3 consecutive readings.

The SHRs and WKY rats were euthanized under general anesthesia by an intraperitoneal injection of pentobarbital sodium (50 mg/kg). The kidneys and spleens were harvested, weighed and fixed in formalin. After 72 h, the samples were dehydrated, embedded in paraffin, and sliced into 5-μm-thick sections. These sections were mounted on slides and stained with hematoxylin and eosin (H&E). The slides were photographed under a light microscope (Olympus, Tokyo, Japan). The damaged tubules were identified based on the presence and severity of the renal tubular epithelial cell degeneration, necrosis, loss of brush border cells, intraluminal casts and inflammatory cell infiltration, as assessed by two professional pathologists at 10 high-power fields (magnification, ×400) per section. The percentages of histological changes in the kidney tissues were scored with a semi-quantitative scale, which was designed to evaluate the degree of tubular damage as previously described (18): 0, normal kidney; 1, minimal damage (5% involvement); 2, mild damage (5–25% involvement); 3, moderate damage (25–50% involvement); 4, severe damage (50–75% involvement); and 5, most severe damage (>75% involvement).

The SHR and WKY rats were euthanized by 30 mg/l sodium pentobarbital anesthesia (50 mg/kg, i.p.). Peripheral blood was collected with heparin-coated capillary glass tubes from abdominal aorta and allowed to clot at ambient temperature for 15 min. The blood samples obtained were centrifuged at 800 × g for 10 min at 4°C to obtain serum. ELISA was performed to quantify and compare the cytokine levels (IL-2 and IL-6) in serum according to the manufacturer's instructions with rat platinum ELISA kits. The reaction was measured at 450 nm with a microplate reader (Dynatech, Denkendorf, Germany). A standard curve was prepared from IL-2 and IL-6 standard dilutions before the IL-2 and IL-6 concentrations were determined in the rat samples. The concentration of each cytokine was expressed in pg/ml.

The spleens were harvested from the same rats as those above into a tissue culture dish and used to prepare a single cell suspension by gentle pressing with the plunger of a syringe. The isolated cells were passed through a 200 μM cell strainer (BD Biosciences, San Jose, CA, USA) and collected. The cell suspensions were centrifuged at 200 × g for 10 min at room temperature. The red blood cells were isolated, treated with the erythrocyte lysis buffer, and washed twice with phosphate-buffered saline (PBS). After cell counting with a counting chamber and viability detection by trypan blue exclusion (>95%), they were resuspended in an appropriate volume of PBS to a final concentration of approximately 1×10⁶ cells/ml. The cells were incubated at 4°C for 15 min in the dark with fluorochrome-labelled mAbs for surface antigen markers, including T lymphocytes (CD3⁺), T-helper cells (CD3⁺CD4⁺), cytotoxic T cells (CD3⁺CD8⁺) and regulatory T cells (CD4⁺CD25⁺). The cells were resuspended in 500 μl of PBS. Subsequently, approximately 20,000 cells were analyzed using a flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA), with an initial gate set by forward/side scatter (FSC/SSC) dot plots. Isotype-matched antibodies were used as negative control samples to confirm the specificity of antibody binding and eliminate nonspecific receptor binding or other cellular protein interactions. The proper fluorescence compensation was set to ensure that negative and positive cells were identical and used to gate the population. For the splenic cell suspension, CD45 staining was used to identify leukocytes, in the CD45⁺ gate, T cells were identified by the anti-CD3, -CD4, -CD8 and -CD25 antibodies.

The spleens were harvested from the same SHRs and WKY rats as those above into a tissue culture dish and used to prepare a single cell suspension via the method above. Cell suspension was resuspended in an appropriate volume of PBS to a final concentration of approximately 1×10⁶ cells/ml and fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Tween-20 for 20 min at room temperature. Following incubation in blocking buffer containing 10% goat serum, the cells were incubated with the anti-rat Cx43 antibodies at 4°C for 30 min prior to incubation with the FITC-labeled goat anti-mouse IgG secondary antibody in the dark for 30 min at 4°C with. Negative control samples were incubated with isotype-matched antibodies. The cells were washed twice with PBS and incubated with CD4 or CD8 antibodies for flow cytometry analysis as described above.

Splenic cells from the SHRs and WKY rats were collected in respective centrifuge tube containing the lymphocyte separation medium. After density gradient centrifugation (400 × g for 30 min), the splenic mono-nuclear cells were isolated. The red blood cells were lysed by the addition of the erythrocyte lysis solution for 5 sec before washing the pellet twice with PBS. The pellet was resuspended in 1 ml of RPMI-1640 medium containing 10% FBS, 100 units penicillin and 100 μg/ml streptomycin. Following 3 h of incubation at 37°C in 5% CO₂, non-adherent cells were collected and adjusted to 1×10⁶ cells/ml in the culture medium. The mitogen ConA was used to stimulate the lymphocytes. Furthermore, the Gap27 peptide was used to evaluate the effects on lymphocytes activation and proliferation (Gap27 dissolved in Hanks balanced salt solution was used as gap junctional communication inhibitor). The cells were dispensed into 96-well round-bottomed plates as 100 μl triplicates. The wells were divided into the following 4 treatment groups: the control, ConA, Gap27 and Gap27 + ConA groups. The control group contained cells without stimulation. In the ConA group, the lymphocytes were stimulated with ConA (5 μg/ml) for 24 h. In the Gap27 group, the lymphocytes were stimulated with Gap27 (500 μM) for 48 h. In the Gap27 + ConA group, Gap27 (500 μM) was used to incubate the lymphocytes for 48 h prior to stimulation with ConA (5 μg/ml) for 24 h. The plates were incubated at 37°C under 5% CO₂ in a humidified incubator. After 72 h, the splenic lymphocytes from the SHRs and WKY rats were harvested to determine the cell proliferation with CCK-8 kits according to the manufacturer's instructions. The optical density was measured using a microplate reader (Dynatech), with a test wavelength of 450 nm.

To calculate the frequency of different lymphocyte subpopulations in response to ConA stimulation and treatment with the GJ inhibitor, Gap27, each group of cultured cell suspensions from the WKY rats and SHRs was incubated and stained for 15 min at 4°C in the dark for flow cytometic analysis with the following antibodies: FITC-anti-CD3, APC-anti-CD4, PE-anti-CD8 and PE-anti-CD25. The samples were analyzed with the experimental procedure for flow cytometry as mentioned above. The cell proliferation rate was caclulated as follows: cell proliferation rate = (ConA group-control group)/ConA group ×100%. The cell inhibition rate was caclulated as follows: cell inhibition rate = [ConA group−(Gap27 + ConA) group]/ConA group ×100%

Total RNA from the cultured lymphocytes from the SHRs and WKY rats was extracted using TRI reagent. The RNA concentration and purity were determined by measuring spectrophotometric absorbance at 260 nm (A₂₆₀) and the A₂₆₀/280 ratio with a Nanovue™ spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). A 1 μl aliquot of RNA was used to generate cDNA in 10 μl reaction volumes with the PrimeScript™ RT reagent kit. Reverse transcriptase reactions were operated in a thermocycler (Takara, Mountain view, CA, USA) for 15 min at 37°C, followed by incubation at 85°C for 5 sec. For each diluted cDNA sample (performed in triplicate for each gene), 2 μl of cDNA was amplified in 10 μl reactions with 5 μl of 2X SYBR-Green PCR master mix as gene-specific primers: IL-2 forward, 5′-GCA CCT GTA AGT CCA GCA AC-3′ and reverse primer, 3′-ACG CTT GTC CTC CTT GTC A-5′; IL-6 forward, 5′-TTG GGA CTG ATG TTG TTG AC-3′ and reverse primer, 3′-TGT GGG TGG TAT CCT CTG T-5′; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward, 5′-CTC TCT GCT CCT CCC TGT TC-3′, and reverse primer, 3′-GCC AAA TCC GTT CAC ACC G-5′. The real-time PCR system (Bio-Rad, Hercules, CA, USA) was operated with the following reaction program: 20 sec at 37°C and 2 min at 95°C, followed by 40 cycles of 5 sec at 95°C and 10 sec at 60°C. All assays were done in triplicate and the ΔCq value was computed. A relative quantification assay was performed to evaluate the ratio of each cytokine normalized to the GAPDH expression in each sample.

All results were presented as the means ± SEM for each assessment. All statistical analyses were performed with GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). One-way ANOVA, followed by Tukey's multiple comparison test, was performed to compare more than 3 groups. An unpaired two-tailed t-test was used to establish 2 group comparisons. Differences with p<0.05 were considered statistically significant.

The basal SBP of the WKY rats and SHRs was measured prior to the start of the experiments. At 16-17 weeks of age, the SBP was significantly higher in the SHRs (188.9±3.7 mmHg) than in the age-matched WKY rats (109.3±2.6 mmHg; p<0.01). A tendency toward an increased spleen weight (SW) was observed in the SHRs, although the difference was not significant as compared with the WKY rats. No significant difference was observed in the body weight (BW) or the ratio of the spleen weight to the body weight (SW/BW) ratio between the SHRs and WKY rats (p>0.05) (Table I).

To observe the pathological changes in the target organs resulting from hypertension, we determined the pathological changes in the kidneys and spleens of the SHRs by H&E staining. H&E staining revealed a significant thickening of the vascular wall, inflammatory cell infiltration into part of the renal interstitium, and glomerular atrophy in the kidneys of the SHRs as compared with those of the WKY rats. After blinded semi-quantitative scoring, the tubular damage score in the SHRs (3.1±0.2) was greater than that in the WKY rats (0.7±0.2; p<0.01) (Fig. 1). The SHRs exhibited stenosis in the wall of the central artery in the spleen (Fig. 1).

We evaluated the hypertensive inflammatory response by measuring the concentration of typical Th1 (IL-2) and Th2 (IL-6) cytokines in the serum of the WKY rats and SHRs, as these cytokines are generally regarded as signs of the pro-inflammatory response. The serum concentrations of IL-2 (100.5±15.5 pg/ml) and IL-6 (28.5±1.5 pg/ml) in the SHRs were significantly higher than the IL-2 (50.8±10.0 pg/ml) and IL-6 (22.1±1.0 pg/ml; p<0.05) levels in the WKY rats (Fig. 2). These results suggested that the development of hypertension was accompanied by an inflammatory response and the impairment of kidney and spleen function was caused by inflammation. Simultaneously, the elevated blood pressure may promote lymphocyte activation and the secretion of pro-inflammatory cytokines.

We investigated whether pro-inflammatory lymphocytes can be detected in the spleens of normotensive and hypertensive rats. Flow cytometric analysis (Fig. 3) was used to determine the percentages of CD3⁺ T cells (WKY, 41.8±1.0%; SHR, 41.5±1.6%; p>0.05), CD3⁺CD4⁺ T cells (WKY, 52.5±1.4%; SHR, 54.9±0.7%; p>0.05), CD3⁺CD8⁺ T cells (WKY, 37.2±0.7%; SHR, 36.2±0.9%; p>0.05) and CD4⁺CD25⁺ T cells (WKY, 4.9±0.1%; SHR, 2.6±0.1%; p<0.01), as well as the ratio of CD4⁺/CD8⁺ (WKY, 1.47±0.02; SHR, 1.52±0.03; p>0.05) cells. The frequencies of CD4⁺CD25⁺ T cells were significantly lower in the spleens of SHRs as compared with those of the WKY rats. The percentage of other T cell subtypes, including the CD3⁺, CD3⁺CD4⁺, and CD3⁺CD8⁺ T cells in the spleen and the CD4⁺/CD8⁺ ratio did not differ significantly between the SHRs and WKY rats. These results demonstrated that a population of Tregs may be required for hypertension.

After analyzing the phenotypic characterization of lymphocyte subsets, we compared Cx43 surface expression in splenic lymphocyte subsets between the SHRs and WKY rats. A representative scatter plot in Fig. 4 shows the percentage of CD4⁺Cx43 and CD8⁺Cx43 cells in the spleens from the SHRs and WKY rats. Flow cytometric analysis revealed the absence of significant statistical differences in the percentage of CD4⁺Cx43 or CD8⁺Cx43 in the spleens between the SHRs and WKY rats. However, Cx43 surface expression could not be detected in the SHRs and WKY rats due to the low percentage of CD4⁺CD25⁺ T cells (Fig. 4).

To confirm the involvement of GJs in hypertensive inflammation responses, cultured splenic lymphocytes from the SHRs and WKY rats were treated with ConA (5 μg/ml) alone or ConA for 24 h following treatment with the Cx43 mimetic peptide, Gap27 (500 μM) for 48 h. The activation of lymphocytes in vitro was determined by CCK-8 assay. Statistical analysis of the results is shown in Fig. 5. In the lymphocytes from the WKY rats, compared with the control group (0.53±0.01), significant lymphocyte proliferation was observed in the ConA group (0.59±0.01; p<0.05). Compared to the ConA group, lymphocyte proliferation was significantly decreased in the Gap27 group (0.52±0.01) and Gap27 + ConA group (0.54±0.01; p<0.05). No statistically significant differences were noted between the Gap27 + ConA and control groups. In the lymphocytes from the SHRs, significant lymphocyte proliferation was observed in the ConA group (0.62±0.01) as compared with the control group (0.55±0.01; p<0.05). The lymphocytes in the Gap27 group (0.54±0.01) and Gap27 + ConA group (0.54±0.02) exhibited a significantly decreased proliferation as compared with the ConA group (p<0.05). By contrast, lymphocyte proliferation following Gap27 intervention was similar to that of the control group even after ConA stimulation (p>0.05) (Fig. 5). These data demonstrated that the proliferation of T lymphocytes may be directly regulated by GJs during essential hypertension.

The percentage of T cell subsets was determined for the SHRs and WKY rats treated with Gap27. In the lymphocytes from the WKY rats, the percentages of total T cells (CD3⁺) and CD4⁺CD25⁺ T cells differed significantly among the 4 groups. The percentages of CD3⁺ and CD4⁺CD25⁺ T cells were higher in the ConA-treated lymphocytes than in the control group lymphocytes (p<0.05) (Fig. 6). Under the effects of Gap27, the percentages of CD3⁺ and CD4⁺CD25⁺ T cells in the Gap27 and Gap27 + ConA groups were significantly decreased compared to those of the ConA group (p<0.05). However, the percentages of CD3⁺ and CD4⁺CD25⁺ T cells did not differ significantly between the Gap27 + ConA and control groups. More specifically, the CD4⁺/CD8⁺ ratio was statistically similar in all comparisons (Fig. 6). In the lymphocytes from the SHRs, the trends for the percentages of CD3⁺ and CD4⁺CD25⁺ T cells, as well as the CD4⁺/CD8⁺ ratio, among the 4 groups were consistent with those of the WKY rats (Fig. 6). In addition, the proliferation rates of the CD3⁺ and CD4⁺CD25⁺ T cells, as well as the inhibition rate of the CD4⁺CD25⁺ T cells in the lymphocytes from the SHRs were higher than those of the WKY rats (p<0.01) (Fig. 7).

The blocking of direct intercellular communications among adjacent lymphocytes suppresses the synthesis and release of pro-inflammatory cytokines, such as IFN-γ and IL-2 (17,19). Thus, to further investigate involvement of Cx43-mediated GJIC in the synthesis of pro-inflammatory cytokines in changed T cells subsets during hypertension, the IL-2 and IL-6 cytokine levels were detected following intervention with Gap27 by RT-qPCR. The results obtained fro the 4 groups are presented in Table II. In the SHRs and WKY rats, the IL-2 and IL-6 mRNA expression levels were significantly increased following ConA stimulation (p<0.05). However, the IL-2 and IL-6 mRNA expression levels decreased following treatment with Gap27 in the Gap27 and ConA + Gap27 groups (p<0.05). Notably, the IL-2 and IL-6 mRNA expression levels did not differ significantly between the Gap27 + ConA and control groups (Table II). In addition, the proliferation rate of the lymphocytes from the SHRs expressing IL-2 and IL-6 was higher than that of those from the WKY rats (p<0.05) (Fig. 8). Compared with the lymphocytes from the WKY rats, the Gap27 + ConA group lymphocytes from the SHRs exhibited a significant decrease in IL-2 and IL-6 mRNA expression compared with the ConA group (Table II), and thus the inhibition rate of lymphocytes expressing IL-2 and IL-6 was significantly higher in the lymphocytes from the SHRs than in those from the WKY rats (p<0.01) (Fig. 8). This trend may be explained by the obvious responsiveness of these cytokines to ConA, such that cytokine secretion was immediately inhibited by Gap27. The effects of Gap27 also differed in the lymphocytes obtained from the hypertensive and normotensive rats.