Stimulation of lymphocyte responses by angiotensin II promotes kidney injury in hypertension

Animals.

Wild-type 129/SvEv mice were purchased from Taconic and Agtr1a^−/− mice lacking AT_1A receptors for ANG II were generated as previously described (29). All mice were maintained in the animal facility of the Durham Veterans Affairs Medical Center under local and National Institutes of Health guidelines. All experiments described in this manuscript were included in an animal use protocol approved by the Durham VA Medical Center Institutional Animal Care and Use Committee. These studies used 2- to 4-mo-old male mice.

Model of ANG II-induced hypertension.

Wild-type 129/SvEv mice first underwent left nephrectomy and ANG II (1,000 ng·kg⁻¹·day⁻¹; Sigma) or vehicle (0.9% NaCl, n = 5) was infused continuously with subcutaneous osmotic minipumps (2004, Alzet) for 28 days as previously described (12, 35). Animals received a 6% NaCl diet (Harlan Teklad) during the ANG II infusion period to accentuate hypertension and kidney injury. To examine the contribution of the immune response to hypertensive kidney injury, the ANG II-infused mice were treated with mycophenolate mofetil (MMF; Roche; 100 mg·kg⁻¹·day⁻¹) or vehicle given by gavage beginning 1 day before and continuing throughout the 28-day ANG II infusion period (n ≥ 13 per group). This dose of MMF causes potent immunosuppression in mice without measurable toxicity (30, 46, 58).

Physiological assessments.

Systolic blood pressure was determined in conscious mice by the noninvasive computerized tail-cuff method as previously described (18). Animals underwent 2 wk of training before the initiation of recordings. Blood pressures were measured for 1 wk at baseline, after unilateral nephrectomy, and during 3 wk of ANG II infusion. During weeks 2 and 4 of ANG II infusion, the mice were placed in metabolic cages, and urine was collected for 24 h. Urinary concentrations of albumin were measured in individual samples using a specific ELISA for mouse albumin (Exocell, Philadelphia, PA) as previously described (18). Creatinine concentrations were measured using a picric acid-based method using a kit (Exocell). Albumin excretion is expressed as micrograms per milligram of creatinine. To measure generation of reactive oxygen species in the kidney, we quantitated urinary excretion of 8-isoprostane (32) per the manufacturer's instructions (8-Isoprostane EIA Kit, Cayman Chemical, Ann Arbor, MI). Isoprostane excretion is expressed as picograms per 24 hours.

Histopathologic analysis.

Following 28 days of ANG II infusion, hearts and kidneys were harvested, weighed, and fixed in formalin, sectioned, and stained with Masson trichrome. All of the tissues were examined by a pathologist (P.R.) without knowledge of the treatment groups. The pathological abnormalities were graded based on the presence and severity of component abnormalities including glomerulosclerosis, chronic inflammation, tubular atrophy or casts, fibrosis, and vascular injury. Grading for each component was performed using a semiquantitative scale as previously described (53, 54) where 0 was no abnormality and where 1, 2, 3, and 4 represented mild, moderate, moderately severe, and severe abnormalities, respectively. The total injury score for each kidney was a summation of these component injury scores. Percent glomerulosclerosis was defined as number of glomeruli with evidence of sclerosis divided by the total number of glomeruli in the section.

To assess T lymphocyte infiltration in the kidneys, paraffin-embedded sections were stained with anti-CD3 (clone SP7) per the manufacturer's instructions (Lab Vision, Fremont, CA). For an assessment of the CD4⁺ and CD8⁺ T cell subsets, frozen sections were stained with anti-CD4 (clone RM4–5, catalog no. 550280, BD Biosciences/Pharmingen, San Diego, CA) or anti-CD8 (clone 53–6.7, catalog no. 550281, BD Biosciences/Pharmingen) as previously described (60). On each section, 20 randomly selected fields were then scored in a blinded fashion for the presence or absence of T cell infiltrates (3 or more T cells in field). To quantify the extent of vascular inflammation, vessels were identified on the sections and the severity of perivascular T cell infiltrates was scored based on a previously established method (16) by assigning vessels to quartiles: Normal, no T cells were present; Minimal, 1–4 infiltrating T cells; Moderate, infiltrates containing 5–10 T cells; and Severe, infiltrates with more than 10 T cells.

Quantification of cardiac mRNA expression.

Hearts were harvested, and total RNA was isolated by using an RNeasy Fibrous Tissue Mini Kit per the manufacturer's instructions (Qiagen, Valencia, CA). The gene expression levels of brain natriuretic peptide (BNP) and β-MHC in cardiac tissue were determined by real-time quantitative RT-PCR as previously described (33).

RNase protection assays.

Total cellular RNA was extracted from harvested kidneys using the RNeasy kit (Qiagen), according to manufacturer's instructions, and was stored in RNase-free water at −70°C. To detect cytokine mRNA, a commercially available multiprobe template set (MCK-3b; BD Biosciences, Bedford, MA) was labeled with [α-32P]UTP (PerkinElmer), according to the manufacturer's instructions, and then diluted to a concentration of 270,000 cpm/μl of hybridization buffer. All reagents used in probe synthesis were obtained from BD Biosciences (In Vitro Transcription Kit, catalog 45004K). RNase protection assays were performed using the RNase Protection Assay Kit (BD Biosciences; catalog 45014K) following the manufacturer's protocol. Gels from the assay were dried under vacuum at 80°C for 60 min and then were placed on film in a cassette with an intensifying screen and developed at −70°C. Films were scanned and bands were analyzed as a ratio of target RNA/L32 control using the Scion Image for Windows program.

Quantitation of cytoskeletal rearrangements.

To provide a quantitative assessment of actin polymerization in lymphocytes, F-actin formation was determined using flow cytometry. Mouse splenocytes were isolated as previously described (45) and were stimulated with ANG II (1 μM) or anti-CD3 (1 μg/ml) in the presence or the absence of losartan (10 μM, Merck, Whitehouse Station, NJ) or Rho kinase inhibitor Y-27632 (10 μM, Calbiochem). The cells were harvested, fixed, and permeabilized with a formaldehyde/saponin solution (Cytofix, BD Biosciences) and stained with an Alexafluor 488-conjugated phalloidin (Molecular Probes). The cells were then analyzed by flow cytometry, and the percentage of polymerized actin was determined by comparing the percent change in mean channel fluorescence in the activated vs. unstimulated cells (17). Results shown reflect the average of at least three separate experiments. In some of the actin polymerization experiments, pure populations of splenic T cells were isolated using a Pan T Cell Isolation Kit (Miltenyi Biotec, Auburn, CA). Purity of the cell population was confirmed by fluorocytometry and averaged 98%.

Statistical methods.

The values for each parameter within a group were expressed as means ± SE. For comparisons between groups with normally distributed data, statistical significance was assessed using ANOVA followed by unpaired t-test. For comparisons between groups with nonnormally distributed data, the Mann Whitney U-test was employed. For comparisons within groups, normally distributed variables were analyzed by a paired t-test, whereas nonnormally distributed variables were analyzed by the Wilcoxon signed rank test. Chi square analysis was used for analysis of the frequencies of perivascular T cell infiltrates in kidneys.

Immunosuppression with MMF does not alter the hypertensive response to chronic ANG II infusion and high salt feeding.

To examine the role of lymphocyte responses in ANG II-dependent hypertension, we infused uninephrectomized mice with ANG II for 4 wk while administering MMF (100 mg/kg) or its vehicle daily by gavage. The combination of ANG II infusion and high salt diet caused marked hypertension and the severity of blood pressure elevation was not significantly affected by MMF (MMF 214 ± 11 vs. vehicle 206 ± 8 mmHg; P = NS; Fig. 1A).

MMF does not attenuate cardiac hypertrophy in ANG II-dependent hypertension.

In vehicle-treated animals, ANG II and high salt feeding caused marked cardiac hypertrophy (heart wt:body wt ratio 9.8 ± 0.8 mg/g) compared with high salt diet alone (6.5 ± 0.2 mg/g; P < 0.02; Fig. 1B) or normal diet (5.4 ± 0.1 mg/g; P < 0.007). On a molecular level, ANG II induced prominent gene expression for markers of cardiac hypertrophy including BNP [18.5 ± 5.1 arbitrary units (au); Fig. 1C] and β-myosin heavy chain (β-MHC; 7.9 ± 1.8 au; Fig. 1D). Treatment with MMF during ANG II infusion had no effect on cardiac hypertrophy (heart wt:body wt ratio 9.0 ± 0.8 mg/g; P = NS; Fig. 1B) or on expression of BNP (17.5 ± 6.4 au; P = NS; Fig. 1C) or β-MHC (9.1 ± 3.6 au; P = NS; Fig. 1D). The finding of equivalent cardiac hypertrophy in vehicle- and MMF-treated groups is consistent with their similar blood pressures and suggests that immune system activation does not make a critical contribution to left ventricular hypertrophy caused by ANG II.

Immunosuppression reduces ANG II-dependent kidney injury.

As a functional measure of the extent of kidney injury, we measured urinary albumin excretion in the experimental groups. ANG II infusion and high salt diet caused marked albuminuria in the vehicle-treated animals at 2 (7,705 ± 1,581 μg albumin/mg creatinine) and 4 wk (10,390 ± 2,324 μg albumin/mg Cr). Despite its lack of effect on blood pressure, administration of MMF reduced urinary albumin excretion by over 60% (Fig. 2A) after 2 (2,528 ± 449 μg albumin/mg Cr; P < 0.008 vs. vehicle) and 4 wk (4,058 ± 1,212 μg albumin/mg Cr; P < 0.04 vs. vehicle).

After 4 wk of ANG II infusion and high salt feeding, kidneys from the vehicle-treated group exhibited significant pathology including glomerulosclerosis, proteinacious casts in renal tubules, robust inflammatory cell infiltration, and interstitial fibrosis (Fig. 2B). Treatment with MMF significantly ameliorated kidney injury reducing the severity of the overall pathology score from 11.5 ± 0.9 to 7.8 ± 0.7 au (P = 0.003; Fig. 2, B–C, Table 1). This included effects of MMF therapy to reduce glomerulosclerosis (18.4 ± 3.0 vs. 29.9 ± 3.2%; P < 0.02; Fig. 2D) and cast formation (1.3 ± 0.2 vs. 2.1 ± 0.2 au; P < 0.008; Fig. 2, E–F). Within the renal interstitium, MMF significantly attenuated inflammatory cell infiltration (1.9 ± 0.2 vs. 3.2 ± 0.4 au; P = 0.006; Fig. 2, B–C) and interstitial fibrosis (0.2 ± 0.1 vs. 0.7 ± 0.2 au; P = 0.03; Fig. 2, B–C). In contrast to negligible consequences in the heart, these data suggest a significant role for immune activation in ANG II-dependent kidney injury.

Activation of intrarenal lymphocyte responses promotes hypertensive kidney injury.

We used immunostaining to examine the surface phenotype of cells infiltrating kidneys from the ANG II-infused mice (Fig. 2B). As shown in Fig. 3B, we found that CD3⁺ cells, presumably T lymphocytes, are a prominent component of the mononuclear cell infiltrates in the kidneys from vehicle-treated mice infused with ANG II. T cells were particularly concentrated in perivascular regions (Fig. 3E) and 83% of vessels from kidneys of mice receiving ANG II infusion and vehicle had moderate to severe perivascular T cell infiltrates. Treatment with MMF caused a dramatic diminution of these T cell infiltrates (Fig. 3, C–D, F–G). This was manifested by a substantial reduction in the frequency of renal vessels with moderate to severe T cell infiltrates to only 28% (P < 0.0001 vs. vehicle; Fig. 3F).

To determine which T cell subsets infiltrated the kidneys of the ANG II-infused animals, we stained kidney sections for CD4 and CD8. ANG II infusion caused a dramatic accumulation of CD4⁺ T helper lymphocytes in the kidney (Fig. 3, H and J), an effect almost completely reversed by treatment with MMF (Fig. 3, I–J). By contrast, although ANG II caused a numerical increase in the fields containing CD8⁺ cells, this increase was not significant compared with the control group or the group treated with ANG II plus MMF (Fig. 3J). As CD8⁺ cells are capable of eliciting reactive oxygen species (ROS) (39), we quantitated renal generation of ROS in the experimental groups by measuring urinary 8-isoprostane excretion. ANG II induced a brisk generation of ROS that was not altered by treatment with MMF (Fig. 3K), indicating that induction of ROS by ANG II depends on cell lineages other than CD8⁺ cytotoxic T cells.

To further assess the effects of MMF on the intrarenal inflammatory response, we examined mRNA expression for key inflammatory cytokine genes. In control mice fed high salt without ANG II infusion, renal expression of inflammatory cytokines IFN-γ, TNF-α, and MIF and profibrotic cytokine TGF-β was negligible (data not shown). Chronic infusion of ANG II prominently increased intrarenal gene expression for this entire panel of cytokines. MMF therapy dramatically attenuated expression of IFN-γ and TNF-α but did not alter the enhanced expression of MIF, suggesting some specificity of the response (Fig. 4, A–B). It has been suggested that ANG II may directly stimulate intrarenal production of the profibrotic molecule TGF-β (31) which may then contribute to the development of kidney fibrosis. Indeed, expression of TGF-β was significantly upregulated in kidneys from ANG II-infused animals. However, MMF dramatically reduced TGF-β mRNA levels suggesting complex control of TGF-β expression in this setting (Fig. 4, A–B).

ANG II stimulates cytoskeletal rearrangements in T lymphocytes.

Our findings from experiments in intact animals suggest that one of the pathways contributing to kidney injury in ANG II-dependent hypertension involves accumulation of T cells around renal vasculature and enhanced expression of cytokines that promote inflammation and fibrosis. In T cells, rearrangements of the actin-myosin cytoskeleton are crucial to the development of full activation and development of the immunological synapse (1, 8, 50). Because ANG II triggers cytoskeletal changes in vascular tissues (27, 34, 55) and in glomerular epithelial cells (52), we considered the possibility that another pathway for control of T cell activation by ANG II might involve effects on actin polymerization.

To test this hypothesis, murine splenocytes were stimulated with ANG II (1 μM) and subsequently stained with a fluorescent phalloidin that binds the polymerized or “filamentous” form of actin (F-actin). We then performed flow cytometry to measure ANG II-dependent F-actin formation as reflected by mean channel fluorescence in wild-type and AT_1A receptor-deficient (Agtr1a^−/−) lymphocytes. Using this assay, we found that ANG II induced marked actin polymerization in wild-type lymphocytes (27.9 ± 8.4%; P = 0.01 vs. vehicle) but did not stimulate F-actin formation in splenocytes from Agtr1a^−/− mice (−16.3 ± 7.5%; P = 0.003 vs. wild-type). Consistent with the notion that cytoskeletal rearrangements facilitate T lymphocyte activation, we found that T cell receptor stimulation with anti-CD3 (1 μg/ml) caused significant actin polymerization in wild-type lymphocytes (28.5 ± 4.0%). This effect was significantly blunted in Agtr1a^−/− lymphocytes (11.5 ± 3.0%; P = 0.007). Furthermore, activation of highly enriched wild-type T lymphocytes with anti-CD3 and anti-CD28 (2 μg/ml) triggered marked actin polymerization, an effect significantly attenuated by coincubation with the ARB losartan (38.9 ± 8.4 vs. 13.3 ± 7.0%; P = 0.009). Thus, ANG II stimulates cytoskeletal rearrangements in T lymphocytes through activation of the AT₁ receptor.

We next explored the mechanism through which ANG II modulates the cytoskeleton in lymphocytes. In vascular smooth muscle cells, ANG II influences actin-myosin cytoskeletal rearrangements via pathways involving Rho-GTPases and their distal effectors such as Rho Kinase; these actions may promote vascular injury (34). We considered the possibility that similar pathways might be responsible for the effects of ANG II to modulate the actin cytoskeleton in lymphocytes. To test this possibility, splenic lymphocytes were exposed to 1 μM ANG II in the presence or absence of the specific Rho kinase inhibitor, Y-27632 (10 μM). As before, exposure to ANG II stimulated F-actin formation (17.1 ± 6.8%) in lymphocytes and this was completely abrogated by Y-27632 (−3.0 ± 5.8%; P < 0.05 vs. ANG II alone). Thus, by stimulating AT₁ receptors on T lymphocytes, ANG II promotes cytoskeletal rearrangements through a pathway requiring Rho kinase.