Role of angiotensin II type 1a receptor in renal injury induced by deoxycorticosterone acetate-salt hypertension.

The aim of this study was to investigate the in vivo role of angiotensin II type 1a (AT1a) receptor in renal damage as a result of hypertension by using transgenic mice with AT1a receptor gene disruption. Transgenic mice that express human liver-type fatty acid binding protein (L-FABP) with or without disruption of the AT1a receptor gene (L-FABP+/- AT1a-/-, and L-FABP+/- AT1a+/+, respectively) were used with urinary L-FABP as an indicator of tubulointerstitial damage. Those female mice were administered subcutaneously deoxycorticosterone acetate (DOCA)-salt tablets plus drinking water that contained 1% saline for 28 d after uninephrectomy. In L-FABP+/- AT1a+/+ mice that received DOCA-salt treatment, hypertension was induced and slight expansion of glomerular area, glomerular sclerosis, and tubulointerstitial damage were observed. In L-FABP+/- AT1a-/- mice that received DOCA-salt treatment, hypertension was similarly induced and the degree of glomerular damage was significantly more severe than in L-FABP+/- AT1a+/+-DOCA mice. Urinary L-FABP levels were significantly higher in L-FABP+/- AT1a-/--DOCA mice compared with those in L-FABP+/- AT1a+/+-DOCA mice. Hydralazine treatment significantly attenuated renal damage that was found in L-FABP+/- AT1a-/--DOCA mice along with a reduction in blood pressure. In summary, activation of the AT1a receptor may contribute to maintenance of the glomerular structure against hypertensive renal damage.-Hisamichi, M., Kamijo-Ikemori, A., Sugaya, T., Ichikawa, D., Natsuki, T., Hoshino, S., Kimura, K., Shibagaki, Y. Role of angiotensin II type 1a receptor in renal injury induced by deoxycorticosterone acetate-salt hypertension.

Angiotensin II (Ang II) type 1 (AT1) receptors exist as 2 isoforms, designated AT1a and AT1b (1), in rodents. AT1a receptor is expressed in podocytes, mesangial cells, renal tubule, and vascular smooth muscle cells (VSMCs), including afferent and efferent arterioles. AT1a receptor plays a major role in the renal actions of Ang II (2–4) and is the murine homolog of the single human AT1 receptor (5).

Although Ang II is widely known to be an aggravating factor in renal disease, activation of AT1a receptor has recently been reported to be involved in the myogenic response of vascular smooth muscle in the autoregulatory system of renal blood flow, which is important for protecting the glomerular capillaries against rise in arterial pressure (6, 7). Furthermore, a previous report by Inokuchi et al. (8) showed that disruption of the AT1a receptor gene led to a lack of interaction between mesangial cells and the glomerular basement membrane (GBM), which indicated that signaling via the AT1a receptor plays an important role in maintaining the glomerular structure. Those results suggested that hypertension may impair the glomerular structure as a result of absence of myogenic control by constriction of afferent arterioles or loss of interconnection between the mesangial cells and GBM. Consequently, transgenic mice with AT1a receptor gene disruption may endure a greater degree of renal damage under hypertensive conditions.

Progression of renal disease is more correlated with the degree of tubulointerstitial damage compared with that of glomerular damage (9–11). Monitoring tubulointerstitial damage is important in the prevention of progression of renal disease. Liver-type fatty acid binding protein (L-FABP) is a small molecular mass protein of 14 kDa that is expressed in human proximal tubules of the kidneys. In various renal diseases, increase in urinary excretion of L-FABP accurately reflects the degree of tubulointerstitial damage; therefore, urinary L-FABP was approved in 2011 by the Ministry of Health, Labor, and Welfare in Japan for use as a tubular injury biomarker in clinical practice.

In this study, we investigated the effect of hypertension on both the glomerulus and the tubulointerstitium in transgenic female mice with disruption of the AT1a receptor gene to confirm the in vivo action of the AT1a receptor in the maintenance of the renal structure in the deoxycorticosterone acetate (DOCA)–salt hypertension model, which is widely used in hypertension research. To use urinary L-FABP as an indicator of tubulointerstitial damage, the human L-FABP gene was inserted into the genome of transgenic mice in addition to disruption of the AT1a receptor gene because L-FABP is not expressed in the kidney of wild-type mice.

MATERIALS AND METHODS

Animals

Studies were conducted in accordance with the St. Marianna University School of Medicine Institutional Guide for Animal Experiments and the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA). Human L-FABP chromosomal transgenic mice of C57/BL6 background (L-FABP+/−AT1a+/+; patent WO0073791) (12) and AT1a receptor-knockout mice (L-FABP−/−AT1a−/−) of C57/BL6 background were generated as previously described (13). L-FABP+/−AT1a+/+ and L-FABP−/−AT1a−/− mice were interbred, and transgenic (L-FABP+/−AT1a+/−) mice that were heterozygous for AT1a gene ablation and L-FABP expression were obtained. After backcrossing of these mice, L-FABP+/−AT1a−/− animals were obtained.

In a preliminary study, approximately 70% of male mice died within 28 d after DOCA-salt administration. Therefore, 7- to 8-wk-old female L-FABP+/−AT1a+/+ (n = 49) and L-FABP+/−AT1a−/− mice (n = 34) were used for experiments.

Experimental design

All L-FABP+/−AT1a+/+ and L-FABP+/−AT1a−/− mice were subjected to left nephrectomy under inhalation anesthesia with 2% isoflurane. One week after uninephrectomy, L-FABP+/−AT1a+/+ and L-FABP+/−AT1a−/− mice were divided into 3 groups. The DOCA-salt hypertension group (L-FABP+/−AT1a+/+-DOCA, n = 22; L-FABP+/−AT1a−/−-DOCA, n = 11) received systemic DOCA (75 mg/mouse) using tablets (SM-121; Innovative Research of America, Sarasota, FL, USA) implanted into the dorsal subcutaneous tissue under isoflurane anesthesia and were provided with drinking water that contained 1% NaCl for 28 d. Antihypertensive treatment groups [L-FABP+/−AT1a+/+-DOCA + hydralazine (Hyd), n = 9; L-FABP+/−AT1a−/−-DOCA+Hyd, n = 14] were administered hydralazine at a dose of 4 mg/kg/d in 1% NaCl water in addition to DOCA and salt water, whereas control mice (L-FABP+/−AT1a+/+ control group, n = 18; L-FABP+/−AT1a−/− control group, n = 9) underwent sham operation and were provided with only tap water.

Before these experiments, we confirmed that both systolic blood pressure (SBP; Supplemental Fig. 1) and degree of renal damage (Supplemental Fig. 2) in L-FABP transgenic mice that received DOCA-salt were similar to those in wild-type mice and that the degree of glomerular damage was significantly more severe in the L-FABP+/−AT1a−/−-DOCA group than in the L-FABP+/−AT1a+/+-DOCA group in the preliminary study. Therefore, we considered that the expression of human L-FABP in the proximal tubules would not affect the results of the present study performed using human L-FABP transgenic mice.

On d 28 of treatment, blood was drawn from the inferior vena cava of all mice, and right kidneys were removed while mice were under intraperitoneal anesthesia with pentobarbital at 75 mg/kg/body weight. A part of the right kidney was snap-frozen in liquid nitrogen for protein and mRNA analyses, whereas the other part was fixed in 10% buffered formalin (Wako Pure Chemical Industries, Osaka, Japan) or methyl carnoy solution. Small pieces of the right kidney were also fixed in 2% glutaraldehyde for pathologic analysis using electron microscopy.

Blood pressure

Blood pressure was measured in conscious, restrained mice via a tail-cuff apparatus (Softron BP-98A; Softron, Tokyo, Japan) (14). SBP values were derived from an average of 3 measurements per animal at each time point: were 0 (baseline), 14, and 28 d of DOCA-salt administration.

Serum and urinary biochemistry

For urine collection, all mice were housed overnight, individually, in metallic cages with free access to 1% NaCl water or untreated tap water on d 27. Sediment was removed from urine samples by centrifugation at 15,000 rpm for 5 min.

Serum and urinary creatinine levels were measured by using an enzymatic method (Nescoat VLII CRE; Alfresa Pharma, Osaka, Japan) (14). Creatinine clearance was calculated on the basis of serum and urine creatinine concentrations and 24-h urine output. Urinary parameters were reported as ratios relative to urinary creatinine levels. Albuminuria was determined by using the Albuwell assay (Exocell, Philadelphia, PA, USA). Urinary L-FABP was measured by using a 2-step sandwich ELISA (L-FABP ELISA kit; CMIC, Tokyo, Japan) (12). Urinary kidney injury molecule-1 (KIM-1) was measured by using the mouse TIM/Kim-1 ELISA kit (R&D Systems, Minneapolis, MN, USA).

Renal histologic and morphometric analyses

For light microscopic analysis, kidneys were dehydrated and embedded in paraffin. Serial sections (2 μm thick) were obtained for conventional histologic assessment, such as periodic acid–Schiff staining (PAS), and for immunohistochemistry. Evaluations were based on chronic tubulointerstitial injury, glomerular hypertrophy, mesangial expansion, and glomerulosclerosis.

Tubulointerstitial injury was categorized as tubular dilation with epithelial atrophy and extracellular matrix accumulation in tissue sections stained with PAS. Twenty nonoverlapping fields from the cortical and outer medullary areas were selected under ×200 magnification (single image field, 725.9 × 546.5 μm), and the regions with tubulointerstitial injury, as well as the entire cortical and outer medullary areas, were measured with the Winroof image analyzer, version 6.1 (Mitani, Tokyo, Japan). The degree of tubulointerstitial injury in each region was expressed as a ratio relative to the entire area. Data presented were shown as the fold-increase or -decrease in evaluation of tubulointerstitial injury in the DOCA or DOCA+Hyd groups compared with the control group.

For evaluation of glomerular change, 30 glomeruli in the cortical area were analyzed. Glomerular size was measured at ×200 magnification by using the Winroof image analyzer and defined as the average area of glomerulus. For quantification of mesangial expansion in each glomerulus, the area of mesangial spread, as well as the size of each glomerulus, were measured with the Winroof image analyzer. The degree of mesangial expansion in each glomerulus was expressed as a ratio relative to the total glomerular area. The grade of glomerulosclerosis in each glomerulus was defined as follows: 0, no findings; 1, 1–50% glomerular area affected (segmental sclerosis); and 2, 50–100% glomerular area affected (global sclerosis). At ×200 magnification, the glomerulosclerosis score—modified from the method used by Taneda et al. (15)—for each mouse was calculated as [(1 × the number of grade 1 glomeruli, %) + (2 × the number of grade 2 glomeruli, %)].

These histologic evaluations were performed in a blinded manner by one observer.

Transmission electron microscopy

To determine the presence of podocyte injury on the glomerulus, transmission electron microscopy (TEM) was performed (16). Small pieces of fixed kidney tissue were immersed in osmium tetroxide solution and embedded in acrylic resin. Ultrathin sections (∼100 nm thick) were prepared by using an LKB Ultrotome V (LKB-Produkter AB, Bromma, Sweden), contrasted with saturated aqueous uranyl acetate and Sato’s lead solution, and viewed with a Jeol 1200EX transmission electron microscope (Jeol, Tokyo, Japan) at 80 kV.

Immunohistologic analysis

Tissues fixed in methyl carnoy solution were embedded in paraffin. An indirect immunoperoxidase method was used to identify the antigens, as previously described (17, 18). Type I and III collagens were identified by using rabbit polyclonal antibodies (Cedarlane Laboratories, Burlington, ON, Canada). Macrophages were identified by using a rat monoclonal antibody against F4/80 (BMA Biomedicals, Augst, Switzerland). Twenty nonoverlapping fields from the cortical and outer medullary areas were selected under ×200 magnification (single image field, 725.9 × 546.5 μm), and positively stained areas for type I and III collagens were measured with Winroof and expressed as ratios to the total cortical and outer medullary areas. Similarly, the degree of macrophage infiltration in the cortical and outer medullary areas was expressed as a ratio of the positively stained area for F4/80 relative to the entire area. Data presented were shown as the fold-increase or -decrease in positively stained area or cells in the DOCA or DOCA+Hyd groups compared with the control group.

To evaluate the fibrosis of glomerulus, type IV collagen was identified by using rabbit polyclonal antibodies (Cedarlane Laboratories) in sections that were fixed in formalin because collagen type IV is a constituent of the mesangial matrix and increases in amount in many forms of glomerular injury (19–21). Thirty glomeruli in the cortical area were analyzed, and positively stained areas for type IV collagen in each glomerulus as well as the size of each glomerulus were measured with Winroof image analyzer and expressed as a ratio relative to the total glomerular area.

Real-time quantitative PCR

Total RNA was extracted from frozen kidneys and reverse transcribed as previously described (17, 18). Real-time quantitative PCR was performed by using a TaqMan Step One Plus system (Thermo Fisher Scientfic, Waltham, MA, USA) to measure mRNA levels of monocyte chemoattractant protein-1 (MCP-1), IL-18, hypoxia-inducible factor-1α (HIF-1α), heme oxygenase-1 (HO-1), TGF-β, connective tissue growth factor (CTGF), plasminogen activator inhibitor-1 (PAI-1), angiotensinogen (AGT), megalin, and glyceraldehyde-3-phosphate dehydrogenase. Expression levels of these transcripts in each sample were normalized to glyceraldehyde-3-phosphate dehydrogenase expression levels and were shown as the fold-increase or -decrease in mRNA expression in the DOCA or DOCA+Hyd groups compared with the control group, with the exception of megalin as there was a significant difference in gene expression of megalin between AT1a+/+-control and AT1a−/−-control groups.

Statistical analysis

All values are expressed as means ± se. Statistical significance was set at P < 0.05. Wilcoxon signed rank test for paired data was used. Differences on d 14 and 28 from d 0 in each group were analyzed by using the Steel test. Six groups were analyzed by Kruskal-Wallis 1-way ANOVA followed by Mann-Whitney U test using JMP 11.2.1 (SAS Institute, Cary, NC, USA).

RESULTS

SBP in response to DOCA-salt

SBP was significantly lower in L-FABP+/−AT1a−/− mice than in L-FABP+/−AT1a+/+ mice on d 0. SBP was significantly lower in the L-FABP+/−AT1a−/− control group than in the L-FABP+/−AT1a+/+ control group during the 28 d of DOCA treatment. SBP was increased on d 14 and 28 of DOCA treatment in the L-FABP+/−AT1a−/− control group compared with baseline, whereas SBP did not increase during the 28 d in the L-FABP+/−AT1a+/+ control group. SBP was similar between the L-FABP+/−AT1a+/+-DOCA and L-FABP+/−AT1a−/−-DOCA groups on d 14 and 28 of DOCA administration, but it was significantly increased on both days compared with their respective controls and baselines (P < 0.05; ). In DOCA-salt hypertensive L-FABP+/−AT1a+/+ mice that received Hyd, although SBP was still significantly higher compared with the control group on d 14, it was significantly lower than that in the untreated L-FABP+/−AT1a+/+-DOCA group on d 14 (P < 0.05). On d 28, the SBP in the L-FABP+/−AT1a+/+-DOCA+Hyd group was significantly lower than that in both the control and DOCA groups (P < 0.05). In DOCA-salt hypertensive L-FABP+/−AT1a−/− mice that received Hyd, although the SBP was still significantly higher than baseline compared with the control group on d 14 and 28, it was significantly lower than that in the untreated L-FABP+/−AT1a−/−-DOCA group on both d 14 and 28 (P < 0.05). SBP was similar between the L-FABP+/−AT1a+/+-DOCA+Hyd and L-FABP+/−AT1a−/−-DOCA+Hyd groups on d 14 and 28.

Figure 1.

Time-related changes in SBP in L-FABP+/−AT1a+/+ (A) and L-FABP+/−AT1a−/− mice (B). Values are means ± se. *P < 0.05 vs. the control group on the same day; †P < 0.05 vs. the same group on d 0; ‡P < 0.05 vs. the AT1a+/+-control group on the same day; §P < 0.05 vs. the AT1a−/−-DOCA group on the same day; #P < 0.05 vs. AT1a+/+-DOCA on the same day; ¶P < 0.05 vs. the AT1a+/+-DOCA+Hyd group on the same day.

Body weight in response to DOCA-salt

Body weight in the L-FABP+/−AT1a+/+-DOCA+Hyd group was significantly greater than that in both the L-FABP+/−AT1a+/+-DOCA and L-FABP+/−AT1a−/− -DOCA+Hyd groups. In all groups except for the L-FABP+/−AT1a+/+-control and L-FABP+/−AT1a+/+-DOCA+Hyd groups, although the body weights were significantly increased on d 28 compared with d 0, the levels were similar between each group on d 28 ().

TABLE 1.

Time-related changes in body weight and serum creatinine levels

Group	Body weight (g)		Creatinine clearance (ml/min/g kidney), d 28
	d 0	d 28
AT1a+/+ control	20.96 ± 0.41	22.42 ± 0.31	0.12 ± 0.02
AT1a+/+ DOCA	19.57 ± 0.33	22.92 ± 0.34*	0.13 ± 0.03
AT1a+/+ DOCA+Hyd	22.22 ± 0.46#,§	23.61 ± 0.55	0.11 ± 0.02
AT1a−/− control	19.60 ± 0.60	22.04 ± 0.43*	0.18 ± 0.04
AT1a−/− DOCA	19.33 ± 0.36	22.73 ± 0.65*	0.26 ± 0.09
AT1a−/− DOCA+Hyd	19.07 ± 0.26	22.75 ± 0.48*	0.20 ± 0.04

Values are means ± se. *P < 0.05 vs. the same group on d 0; #P < 0.05 vs. the AT1a+/+ DOCA groups; §P < 0.05 vs. the AT1a−/− DOCA+Hyd groups.

Creatinine clearance and urinary biochemistry

Although each creatinine clearance level on d 28 in 3 groups of L-FABP+/−AT1a−/− mice tended to be higher than each level in their corresponding group of L-FABP+/−AT1a+/+ mice, there were no significant differences between the 6 groups on d 28 (Table 1).

Urinary albumin levels significantly increased in the L-FABP+/−AT1a+/+-DOCA, L-FABP+/−AT1a+/+-DOCA+Hyd, L-FABP+/−AT1a−/−-DOCA, and L-FABP+/−AT1a−/−-DOCA+Hyd groups on d 28 compared with their respective control group (P < 0.05; ). Levels of the DOCA group were significantly decreased by Hyd administration in both L-FABP+/−AT1a+/+ and L-FABP+/−AT1a−/−mice (P < 0.05). Levels of urinary albumin in the L-FABP+/−AT1a−/−-DOCA and L-FABP+/−AT1a−/−-DOCA+Hyd groups were significantly higher than those in the L-FABP+/−AT1a+/+-DOCA and L-FABP+/−AT1a+/+-DOCA+Hyd groups on d 28 (P < 0.05).

Figure 2.

Urinary albumin (A), urinary human L-FABP (B), and urinary KIM-1 (C) excretions at 28 d after DOCA insertion. Cr, creatinine. Data are reported as means ± se. *P < 0.05 vs. the control group in AT1a+/+; **P < 0.05 vs. the control group in AT1a−/−; #P < 0.05 vs. the AT1a+/+-DOCA group; §P < 0.05 vs. both of the DOCA+Hyd groups; ¶P < 0.05 vs. the AT1a−/−-DOCA group.

Urinary human L-FABP (Fig. 2) and KIM-1 (Fig. 2) levels significantly increased in L-FABP+/−AT1a+/+-DOCA, L-FABP+/−AT1a+/+-DOCA+Hyd, L-FABP+/−AT1a−/−-DOCA, and L-FABP+/−AT1a−/−-DOCA+Hyd groups on d 28 compared with their respective control group (P < 0.05). Their levels in the L-FABP+/−AT1a−/−-DOCA group were significantly higher than those in the L-FABP+/−AT1a+/+-DOCA group, and those were significantly decreased by Hyd treatment (P < 0.05). In the L-FABP+/−AT1a+/+-DOCA+Hyd group, the level tended to be lower compared with the L-FABP+/−AT1a+/+-DOCA group.

Although urinary albumin and human L-FABP levels of the control groups were similar between L-FABP+/−AT1a+/+ and L-FABP+/−AT1a−/−mice, urinary KIM-1 levels were significantly higher in the L-FABP+/−AT1a−/− control than in the L-FABP+/−AT1a+/+ control.

Gene expression in the kidney

mRNA levels of MCP-1 and IL-18 were measured to evaluate the inflammatory response, mRNA level of HIF-1α was determined to evaluate the degree of renal hypoxia, and HO-1 transcript was assessed to evaluate the degree of oxidative stress.

mRNA levels of MCP-1 () were significantly decreased in the L-FABP+/−AT1a−/−-DOCA group compared with the L-FABP+/−AT1a+/+-DOCA group (P < 0.05). mRNA levels of IL-18 (Fig. 3), HIF-1α (Fig. 3), and HO-1 (Fig. 3) were similar between the L-FABP+/−AT1a+/+-DOCA and L-FABP+/−AT1a−/−-DOCA groups. mRNA levels of MCP-1, IL-18, HIF-1α, and HO-1 were significantly increased in both DOCA groups compared with their respective control. Their levels were significantly decreased by Hyd treatment in L-FABP+/−AT1a−/−-DOCA mice (P < 0.05). In L-FABP+/−AT1a+/+-DOCA mice, mRNA levels of only HIF-1α were significantly decreased by treatment. Although the mRNA level of HO-1 was significantly higher in L-FABP+/−AT1a−/−-DOCA+Hyd mice than in L-FABP+/−AT1a+/+-DOCA+Hyd mice, mRNA levels of IL-18 were significantly lower in L-FABP+/−AT1a−/−-DOCA+Hyd mice than in L-FABP+/−AT1a+/+-DOCA+Hyd mice.

Figure 3.

mRNA transcript expression of MCP-1 (A), IL-18 (B), HIF-1α (C), and HO-1 (D). The graph shows the fold-increase or -decease in mRNA expression in the DOCA or DOCA+Hyd groups compared with the control group. *P < 0.05 vs. the AT1a+/+ control group; **P < 0.05 vs. the AT1a−/− control group; #P < 0.05 vs. the AT1a+/+-DOCA group; §P < 0.05 vs. both of the DOCA+Hyd groups; ¶P < 0.05 vs. the AT1a−/−-DOCA group.

Glomerular size (P < 0.05) and expansion of mesangial matrix (P < 0.05) were significantly greater in the L-FABP+/−AT1a−/− control group compared with the L-FABP+/−AT1a+/+ control group.

Glomerular size () was increased in the L-FABP+/−AT1a+/+-DOCA group and was significantly greater in the L-FABP+/−AT1a+/+-DOCA+Hyd group compared with the L-FABP+/−AT1a+/+ control group (P < 0.05). Size was significantly greater in the L-FABP+/−AT1a−/−-DOCA group compared with that of the L-FABP+/−AT1a−/− control and was attenuated by Hyd administration (P < 0.05). Size in the L-FABP+/−AT1a−/−-DOCA+Hyd group was significantly lower than that in the L-FABP+/−AT1a+/+-DOCA+Hyd group (P < 0.05).

Figure 4.

Histologic analyses for glomeruli size, enlargement of mesangial matrix, and presence of glomerular sclerosis in tissues stained with PAS. A) Sizes of glomeruli and the mesangial matrix were increased. B) Presence of glomerular sclerosis. C–E) Sizes of glomeruli (C), enlargement of mesangial matrix (D), and the scores of glomerular sclerosis (E) were assessed quantitatively, as described in Materials and Methods. Values are means ± se. Original magnification, ×200. *P < 0.05 vs. the AT1a+/+ control group; **P < 0.05 vs. the AT1a−/− control group; #P < 0.05 vs. the AT1a+/+-DOCA group; §P < 0.05 vs. both of the DOCA+Hyd groups; ¶P < 0.05 vs. the AT1a−/−-DOCA group.

Expansion of mesangial matrix (P < 0.05; Fig. 4) was increased in the L-FABP+/−AT1a+/+-DOCA group and was significantly greater in the L-FABP+/−AT1a+/+-DOCA+Hyd group compared with the L-FABP+/−AT1a+/+ control group (P < 0.05). Expansion was significantly greater in the L-FABP+/−AT1a−/−-DOCA group compared with that in the L-FABP+/−AT1a−/− control and was attenuated by Hyd administration (P < 0.05). Expansion in the L-FABP+/−AT1a−/−-DOCA+Hyd group was significantly higher than that in the L-FABP+/−AT1a+/+-DOCA+Hyd group (P < 0.05). In L-FABP+/−AT1a+/+ mice, an effect of Hyd on expansion was not observed.

Scores for glomerular sclerosis (P < 0.05; Fig. 4) were significantly greater in both the L-FABP+/−AT1a+/+-DOCA and L-FABP+/−AT1a−/−-DOCA groups compared with their respective controls. Furthermore, scores for these parameters in the L-FABP+/−AT1a−/−-DOCA group were significantly higher than those in the L-FABP+/−AT1a+/+-DOCA group and were reduced by Hyd administration. In L-FABP+/−AT1a+/+ mice, an effect of Hyd on their damage was not observed.

Type IV collagen was immunolocalized to not only the mesangial area of the glomerulus, but also to the tubular basement membrane and interstitium in the kidney (). However, the positively stained area for type IV collagen was quantified and analyzed only in the glomerulus because the degree of renal fibrosis was evaluated by immunohistologic analysis of type I and III collagens. Deposition areas were similar between the L-FABP+/−AT1a+/+-control and L-FABP+/−AT1a−/−-control groups, and were significantly increased in both the L-FABP+/−AT1a+/+-DOCA and L-FABP+/−AT1a−/−-DOCA groups compared with their respective control groups (P < 0.05; Fig. 5). Furthermore, the area in the L-FABP+/−AT1a−/−-DOCA group was significantly greater than that in the L-FABP+/−AT1a+/+-DOCA group and was reduced by Hyd administration. In L-FABP+/−AT1a+/+ mice, an effect of Hyd on their damage was not observed.

Figure 5.

A) Immunohistochemical staining of type IV collagen in the glomerulus. B) The positively stained areas were assessed quantitatively, as described in Materials and Methods. Original magnification, ×200. Values are means ± se. *P < 0.05 vs. the AT1a+/+ control group; **P < 0.05 vs. the AT1a−/− control group; #P < 0.05 vs. the AT1a+/+ DOCA group; ¶P < 0.05 vs. the AT1a−/−-DOCA group.

By TEM (), the foot processes of glomerular epithelial cells were well preserved and the thickness of the GBM was similar in all 6 groups. Podocyte injury was not observed in the L-FABP+/−AT1a+/+-DOCA and L-FABP+/−AT1a−/−-DOCA groups. Mesangial enlargement and a moderate increase in the mesangial matrix were found in the L-FABP+/−AT1a−/− control and L-FABP+/−AT1a−/−-DOCA groups compared with the other groups. In addition, their degrees of increase were greater in the L-FABP+/−AT1a−/−-DOCA group than in the L-FABP+/−AT1a−/− control group, which were attenuated by Hyd administration.

Figure 6.

TEM findings. A) L-FABP+/−AT1a+/+ control group. B) L-FABP+/−AT1a+/+-DOCA group. C) L-FABP+/−AT1a+/+-DOCA+Hyd group. D) L-FABP+/−AT1a−/− control group. E) L-FABP+/−AT1a−/−-DOCA group. F) L-FABP+/−AT1a−/−-DOCA+Hyd. Original magnification, ×2500.

Tubulointerstitial damage, as assessed by PAS staining, was significantly more severe in both the L-FABP+/−AT1a+/+-DOCA and L-FABP+/−AT1a−/−-DOCA groups than in their respective controls, and it was significantly attenuated in the L-FABP+/−AT1a−/−-DOCA group after treatment with Hyd (P < 0.05; ); however, an effect of Hyd was not observed in the L-FABP+/−AT1a+/+-DOCA group. Although there was no significant difference in the degree of tubulointerstitial damage between the L-FABP+/−AT1a+/+-DOCA and L-FABP+/−AT1a−/−-DOCA groups, the degree in the L-FABP+/−AT1a−/−-DOCA+Hyd group was significantly lower compared with the L-FABP+/−AT1a+/+-DOCA+Hyd group.

Figure 7.

A) Histologic analyses for tubulointerstitial damage, macrophage infiltration, and abundance of collagens in tissues stained with PAS. B) Tubulointerstitial damage was assessed quantitatively in PAS-stained sections. C, E, G) Immunohistologic staining for macrophages using an antibody against F4/80 (C), type I collagen (E), and type III collagen (G). D, F, H) Positively stained areas of F4/80 (D), type I collagen (F), and type III collagen (H), were assessed quantitatively, as described in Materials and Methods. The graph shows the fold-increase or -decease in tubulointerstitial damage, macrophage infiltration, and abundance of collagens in the DOCA or DOCA+Hyd group compared with the control group. Original magnification, ×200. *P < 0.05 vs. the AT1a+/+ control group; **P < 0.05 vs. the AT1a−/− control group; #P < 0.05 vs. the AT1a+/+ DOCA group; §P < 0.05 vs. both of the DOCA+Hyd groups; ¶P < 0.05 vs. the AT1a−/−-DOCA group.

Evaluation of macrophage infiltration

Macrophage infiltrates were found in the interstitium in both the L-FABP+/−AT1a+/+-DOCA and L-FABP+/−AT1a−/−-DOCA groups. Level of macrophage infiltration was significantly increased in both the L-FABP+/−AT1a+/+-DOCA and L-FABP+/−AT1a−/−-DOCA groups compared with their respective control groups, and was significantly decreased in the L-FABP+/−AT1a−/−-DOCA group after peripheral vasodilator treatment (P < 0.05; Fig. 7). Such a decrease as a result of treatment was not shown in L-FABP+/−AT1a+/+ mice. Levels of macrophage infiltration in the L-FABP+/−AT1a−/−-DOCA and L-FABP+/−AT1a−/−-DOCA+Hyd groups were significantly lower than those in the respective L-FABP+/−AT1a+/+-DOCA and L-FABP+/−AT1a+/+-DOCA+Hyd groups.

Immunohistologic analysis for type I and III collagens

Areas of type I and III collagen deposition were observed in the interstitium, but not in the glomerulus in our procedure. Areas that were positively stained for type I (P < 0.05; Fig. 7) and type III (P < 0.05; Fig. 7) collagens were significantly increased in both the L-FABP+/−AT1a+/+-DOCA and L-FABP+/−AT1a−/−-DOCA groups compared with their respective control groups, and were significantly decreased in the L-FABP+/−AT1a−/−-DOCA group after Hyd administration. Such a decrease as a result of treatment was not shown in L-FABP+/−AT1a+/+ mice. Although there were no significant differences in the amount of type I and III collagens between the L-FABP+/−AT1a+/+-DOCA and L-FABP+/−AT1a−/−-DOCA groups, their deposit area was significantly lower in the L-FABP+/−AT1a−/−-DOCA+Hyd group than in the L-FABP+/−AT1a+/+-DOCA+Hyd group.

Gene expression related to renal fibrosis in the kidneys

mRNA levels of TGF-β (), CTGF (Fig. 8), and PAI-1 (Fig. 8) were similar between the L-FABP+/−AT1a+/+-DOCA and L-FABP+/−AT1a−/−-DOCA groups. mRNA levels of TGF-β, CTGF, and PAI-1 were significantly increased in both DOCA groups compared with their respective control. Their levels were significantly decreased by Hyd treatment in L-FABP+/−AT1a−/−-DOCA mice (P < 0.05). In L-FABP+/−AT1a+/+-DOCA mice, mRNA levels of only PAI-1 were significantly decreased by treatment. mRNA levels of TGF-β, CTGF, and PAI-1 were significantly lower in L-FABP+/−AT1a−/−-DOCA+Hyd mice than in L-FABP+/−AT1a+/+-DOCA+Hyd mice.

Figure 8.

mRNA transcript expression of TGF-b (A), CTGF (B), and PAI-1 (C). The graph shows the fold-increase or -decease in mRNA expression in the DOCA or DOCA+Hyd groups compared with the control group. *P < 0.05 vs. the AT1a+/+ control group; **P < 0.05 vs. the AT1a−/− control group; #P < 0.05 vs. the AT1a+/+-DOCA group; §P < 0.05 vs. both of the DOCA+Hyd groups; ¶P < 0.05 vs. the AT1a−/−-DOCA group.

Gene expression of AGT in the kidneys

Because renal renin expression is greatly and selectively suppressed in the DOCA-salt model (22–24), the systemic or circulatory renin angiotensin system (RAS) is suppressed in this model. However, the local intrarenal RAS, which is independent of the circulating RAS, is reported to be activated in the DOCA-salt hypertensive model (25). Activation of the local intrarenal RAS is induced by renal production of AGT (26); therefore, gene expression of AGT was evaluated to confirm activation of the local RAS in this study. mRNA levels of AGT in both the L-FABP+/−AT1a+/+-DOCA and L-FABP+/−AT1a−/−-DOCA groups were significantly increased compared with their respective control groups (P < 0.05; ). Although the AGT level did not change as a result of Hyd administration in the L-FABP+/−AT1a+/+-DOCA group, it was significantly decreased by Hyd treatment in the L-FABP+/−AT1a−/−-DOCA group. AGT level in the L-FABP+/−AT1a−/−-DOCA group was similar to that in the L-FABP+/−AT1a+/+-DOCA group.

Figure 9.

AGT mRNA transcript levels on d 28 of DOCA-salt treatment. The graph shows the fold-increase or -decease in AGT mRNA expression in the DOCA or DOCA+Hyd groups compared with the control group. *P < 0.05 vs. the AT1a+/+ control group; **P < 0.05 vs. the AT1a−/− control group; ¶P < 0.05 vs. the AT1a−/−-DOCA group.

Gene expression of megalin in the kidneys

mRNA levels of megalin in the control and DOCA groups of L-FABP+/−AT1a−/− mice were significantly higher compared with levels in the corresponding group of L-FABP+/−AT1a+/+ mice (P < 0.05; ). Megalin levels in both the L-FABP+/−AT1a+/+-DOCA and L-FABP+/−AT1a−/−- DOCA groups were similar to those in their respective control groups. Levels in both the L-FABP+/−AT1a+/+-DOCA+Hyd and L-FABP+/−AT1a−/−-DOCA+Hyd groups were significantly decreased compared with their respective control groups. In L-FABP+/−AT1a−/− mice, megalin level was significantly decreased in the L-FABP+/−AT1a−/−-DOCA+Hyd group compared with the L-FABP+/−AT1a−/−-DOCA group.

Figure 10.

Megalin mRNA transcript levels on d 28 of DOCA-salt treatment. Values are means ± se. *P < 0.05 vs. the AT1a+/+ control group; **P < 0.05 vs. the AT1a−/− control group; #P < 0.05 vs. the AT1a+/+ DOCA group; ¶P < 0.05 vs. the AT1a−/−-DOCA group.

DISCUSSION

The present study revealed that deficiency of the AT1a receptor induced severe glomerular sclerosis compared with AT1a+/+ mice in the DOCA-salt hypertension model and that such glomerular damage was attenuated by a reduction in blood pressure.

Myogenic response, such as contraction of afferent arterioles, plays a more important role in prevention of glomerular damage in hypertensive pathology (27). K.K. showed that the diameters of afferent arterioles in spontaneously hypertensive rats were much smaller than those in Wistar Kyoto rats and that the diameters of efferent arterioles in spontaneously hypertensive rats were larger than those in Wistar Kyoto rats (28), apart from the remnant kidney of subtotal nephrectomy and streptozotocin-induced diabetic kidney, models in which the afferent arteriole was dilated and the efferent arteriole was constricted (29). Myogenic vasoconstriction is induced by depolarization of VSMCs in afferent arterioles after an elevation in transluminal pressure. G-protein-coupled receptors, such as the AT1a receptor, when activated, were reported to function as sensors of membrane stretch in VSMCs that underlie the autoregulatory system (6). It has been reported that activation of the AT1 receptor can be induced by mechanical stress independently from its agonist, Ang II (30), and that AT1a receptor is an Ang II–independent mechanosensor in VSMCs (7). When there is a deficiency in the AT1a receptor, automatic adjustments in the afferent arterioles against hypertension may be lost and systemic hypertension may easily lead to increases in glomerular capillary pressure, which may result in the development of glomerular damage.

In this study, control AT1a−/− and AT1a+/+ mice were subjected to left nephrectomy. Although SBP in control AT1a−/− mice was significantly lower than that in control AT1a+/+ mice during the 28-d study period, it was significantly increased on d 14 and 28 in control AT1a−/− mice. AT1a receptor is known to play an important role in maintenance of basal blood pressure (31). A recent study reported that in mice with VSMC-specific deletion of the AT1a receptor, baseline urinary norepinephrine excretion was significantly higher than that in control mice, and that deletion of the AT1a receptor from VSMCs was associated with enhancement of sympathetic nervous system activity (32). Because deletion of the AT1a receptor augments fluctuations in blood pressure, the SBP in control AT1a−/− mice may have easily increased even after nephrectomy, although no morphologic renal damage was observed on d 14 and 28.

AT1a receptor is expressed in mesangial cells (33) as well as in VSMCs. A previous study reported that the synthesis of the mesangial matrix was partially inhibited and the connection of mesangial cells to the GBM was weak in glomeruli without signaling from the AT1a receptor (8). In our study, loss of connection between mesangial cells and the GBM, in addition to an impaired autoregulatory response of renal blood flow, may have contributed to the severe hypertensive glomerular damage in AT1a−/− mice.

Treatment with an AT1 receptor blocker is strongly recommended for chronic glomerulonephritis or diabetic nephropathy, and progressive immune-mediated renal injury is markedly ameliorated in AT1a−/− mice (34). Inhibition of the AT1a receptor leads to antiproteinuric and anti-inflammatory effects that are independent of the reduction in blood pressure or glomerular capillary pressure (35). Therefore, AT1a receptor deficiency was expected to attenuate renal damage compared with mice with the AT1a receptor; however, this was not demonstrated in our study. Our surprising findings may be a result of the relatively weak glomerular changes that were observed in AT1a+/+ mice that received DOCA-salt treatment. In the Ang II infusion hypertension model (36), it was shown that reduction in AT1a receptor expression did not lead to an improvement in glomerular sclerosis. From these results, it may be difficult to generate severe glomerular injury in other hypertensive renal injury models by using mice with myogenic vasoconstriction (36, 37) for relatively short duration (4 wk). Therefore, it is possible that activation of the AT1a receptor may provide protection only against pressure-dependent glomerular injury.

There was no significant difference in the degree of tubulointerstitial damage between DOCA-salt hypertensive AT1a−/− and AT1a+/+ mice; however, the mechanism by which tubulointerstitial damage was induced by DOCA-salt treatment may be different between the 2 groups. On the one hand, in AT1a+/+ mice that received DOCA-salt treatment, glomerular damage was relatively mild, and increases in renal AGT expression were found. A previous study has shown that activation of intrarenal RAS may play a pivotal role in the control of tubulointerstitial damage (25). On the other hand, in AT1a−/− mice that received DOCA-salt treatment, unfavorable effects of Ang II could not be transmitted via the AT1a receptor to the kidney, which led to an improvement in tubulointerstitial damage along with the amelioration of glomerular damage as a result of a reduction in SBP. Thus, an increase in urinary albumin and a decrease in postglomerular flow caused by severe glomerular damage may be important for the occurrence of tubulointerstitial damage. Furthermore, Ma et al. (38) reported that up-regulation of megalin may be related to acceleration of renal injury as a result of obesity in AT1a−/− mice. In fact, although mRNA levels of megalin did not increase as a result of DOCA administration in either AT1a+/+ or AT1a−/− mice, these levels in the AT1a−/−-control and AT1a−/−-DOCA groups were significantly higher than those in the AT1a+/+-control and AT1a+/+-DOCA groups in this study. Furthermore, levels of urinary tubular marker, urinary KIM-1, at baseline in the AT1a−/−-control group were significantly higher than those in the AT1a+/+-control group, though tubulointerstitial damage did not occur. Although a precise mechanism has not been revealed, the difference in megalin expression may, in part, be related to the progression of tubulointerstitial damage in the AT1a−/−-DOCA group.

In AT1a+/+ mice, increases in urinary albumin, urinary L-FABP, and urinary KIM-1 as a result of DOCA administration—which were significantly lower than those in AT1a−/− mice—were offset by reducing blood pressure. However, renal glomerular and tubulointerstitial damage were not attenuated. This result indicated that, although an increase in systemic blood pressure may have caused glomerular hypertension, which led to an increase in urinary albumin and production of tubular stress even in mice with AT1a receptor, renal histologic damage in AT1a+/+mice was mainly induced by a mechanism other than an increase in blood pressure, such as a change in components of the RAS.

AT1a−/− mice that were used in our study retained expression of Ang II type 1b (AT1b) and type 2 receptors. Gene expression levels of both AT1b and type 2 receptors in the kidneys of DOCA-salt–treated AT1a−/− mice did not increase compared with those of control AT1a−/− mice (Supplemental Fig. 3). There is a possibility that the AT1b receptor has high mechnosensitivity and contributes to myogenic vasoconstriction in VSMCs (39–41). To evaluate the role of AT1b receptor in the DOCA-salt hypertension model, FABP−/−AT1a−/−mice were subjected to left nephrectomy and were administered both DOCA-salt and losartan, which blocks all type 1 angiotensin receptors, during the 28 d period (Supplemental Fig. 4). Increase in blood pressure as a result of DOCA-salt was not reduced by losartan treatment. Although expansion of mesangial matrix and glomerular size induced by DOCA-salt tended to be slightly prevented by losartan treatment, the score for glomerular sclerosis was not attenuated. This result suggested that blocking AT1b receptor in addition to AT1a receptor did not increase vulnerability to pressure-dependent glomerular injury. In the kidney, using AT1a-deficient mice, it has been reported that AT1b receptor expression was primarily concentrated in podocytes and that activation of AT1b receptor caused acceleration of glomerular inflammation with autoimmune disease (42). In the present model, although podocyte injury was not observed by TEM, we considered, using the AT1a deficient mice, that the activation of AT1b receptor contributed slightly to occurrence of glomerular injury in the DOCA-salt hypertension model. Further study is needed to clarify the role of AT1b receptor in the DOCA-salt model. Furthermore, gene expression levels of mineralocorticoid receptor in the kidneys of DOCA-salt–treated AT1a+/+ and DOCA-salt–treated AT1a−/− mice showed no significant differences (data not shown). Therefore, it was speculated that the amount of aldosterone loading in the kidneys was equivalent in the 2 groups.

Although the degree of tubulointerstitial damage in the DOCA-salt–treated AT1a−/− mice was similar to that in AT1a+/+ mice that received DOCA-salt, urinary human L-FABP levels in the former were higher than those in the latter. In clinical practice, urinary L-FABP is a tubular marker that reflects the progression of chronic kidney disease, as demonstrated by significantly higher L-FABP levels in patients with deteriorating renal function as opposed to low levels in those with stable renal function (43). Furthermore, urinary L-FABP was demonstrated to be more sensitive than urinary protein in predicting progression of chronic kidney disease (44). Therefore, in DOCA-salt–treated AT1a−/− mice, further progression of tubulointerstitial impairment was expected.

Regarding the higher body weight in the L-FABP+/−AT1a+/+-DOCA+Hyd group on d 0, there was a possibility that the DOCA load on the kidneys in the L-FABP+/−AT1a+/+-DOCA+Hyd group was less than that in the other DOCA-treated groups. However, the degree of renal histologic damage was not attenuated in the L-FABP+/−AT1a+/+-DOCA+Hyd group. Although glomerulosclerosis was significantly greater in DOCA-salt–treated AT1a−/− mice than in AT1a+/+ mice that received DOCA-salt, expression of genes related to renal fibrosis, such as TGF-β, CTGF, and PAI-1, was similar between those groups and had the same tendency as the deposition levels as those of type I and III collagen in the interstitium. Because total RNA was extracted from frozen kidneys containing considerable amounts of tubulointerstitium, the differences in gene expression in the glomeruli may be disregarded in such a model that incorporates both glomerular and tubulointerstitial damage. Therefore, we considered that the gene expression results reflected the degree of interstitial fibrosis.

In a preliminary study, mortality of male mice after DOCA-salt administration was markedly high. Sex difference in the DOCA-salt hypertension model is well known, and male mice were reported to show more severe hypertension compared with females in the DOCA-salt hypertension model (45). Although the autopsies of DOCA-salt male mice were not performed, cause of death might be speculated to be cardiovascular disease or cerebrovascular disease induced by hypertension.

In summary, genomic deficiency of the AT1a receptor exhibited an increased vulnerability to pressure-dependent renal injury; thus, activation of the AT1a receptor may contribute to the maintenance of the glomerular structure against hypertensive renal damage. The reported limited efficacy of RAS suppression on inhibition of renal dysfunction (46–48) suggests that the AT1a receptor plays an important role in the maintenance of autoregulation of renal blood flow and the glomerular structure against hypertensive pathophysiology.