Enhanced hemodynamic responses to angiotensin II in diabetes are associated with increased expression and activity of AT1 receptors in the afferent arteriole

Abstract

The prevalence of hypertension is about twofold higher in diabetic than in nondiabetic subjects. Hypertension aggravates the progression of diabetic complications, especially diabetic nephropathy. However, the mechanisms for the development of hypertension in diabetes have not been elucidated. We hypothesized that enhanced constrictive responsiveness of renal afferent arterioles (Af-Art) to angiotensin II (ANG II) mediated by ANG II type 1 (AT1) receptors contributes to the development of hypertension in diabetes. In response to an acute bolus intravenous injection of ANG II, alloxan-induced diabetic mice exhibited a higher mean arterial pressure (MAP) (119.1 ± 3.8 vs. 106.2 ± 3.5 mmHg) and a lower renal blood flow (0.25 ± 0.07 vs. 0.52 ± 0.14 ml/min) compared with nondiabetic mice. In response to chronic ANG II infusion, the MAP measured with telemetry increased by 55.8 ± 6.5 mmHg in diabetic mice, but only by 32.3 ± 3.8 mmHg in nondiabetic mice. The mRNA level of AT1 receptor increased by ~10-fold in isolated Af-Art of diabetic mice compared with nondiabetic mice, whereas ANG II type 2 (AT2) receptor expression did not change. The ANG II dose-response curve of the Af-Art was significantly enhanced in diabetic mice. Moreover, the AT1 receptor antagonist, losartan, blocked the ANG II-induced vasoconstriction in both diabetic mice and nondiabetic mice. In conclusion, we found enhanced expression of the AT1 receptor and exaggerated response to ANG II of the Af-Art in diabetes, which may contribute to the increased prevalence of hypertension in diabetes.

diabetes mellitus and hypertension are two major health issues in the United States. The prevalence of diabetes was 9.3% as of 2012, and hypertension affects about one-third of the adult population (69). Diabetes and hypertension are interrelated and frequently coexist (91). The prevalence of hypertension in diabetic subjects is approximately twice that of the nondiabetic population (25, 51, 72, 93, 95). The presence of hypertension in diabetic patients further exacerbates their morbidity and mortality (25, 28, 52). However, the pathophysiological mechanisms for the development of hypertension in diabetes have not been completely elucidated.

The renin-angiotensin system (RAS) plays an essential role in regulating blood pressure and is one of the most important therapeutic targets for hypertension (21, 33, 34). RAS blockers, such as angiotensin-converting enzyme inhibitors and angiotensin receptor blockers, have been recognized as the first-line treatment with additional beneficial effects for hypertensive patients with diabetes, which suggests the involvement of RAS in the development of hypertension in diabetic subjects (4, 50, 82). Although plasma renin was suppressed (18, 27, 77) or normal (11, 61) in diabetic patients, the responsiveness of blood pressure to angiotensin II (ANG II) was demonstrated to be exaggerated in normotensive and mildly hypertensive patients with Type I diabetes (24, 100). However, the mechanism of the enhancement in blood pressure response to ANG II remains elusive.

A previous study with kidney cross-transplantation between wild-type and ANG II type 1 (AT1) receptor-deficient mice examined the action of AT1 receptors in the kidney. They demonstrated the predominant role of the renal AT1 receptors in the development and maintenance of ANG II-dependent hypertension (20). The afferent arterioles (Af-Arts) are the major resistance vessels in the kidney and play a critical role in regulating renal hemodynamics and blood pressure (23, 29, 53, 57). Therefore, we hypothesized in the present study that the increased constrictive responsiveness of the Af-Art to ANG II mediated by the AT1 receptor contributes to the enhanced response of renal hemodynamics to ANG II and the development of hypertension in diabetes. We examined the responses of blood pressure and renal hemodynamic changes to acute and chronic ANG II infusion in diabetic mice and measured the ANG II dose-response curve in the isolated perfused Af-Art.

METHODS

All studies were approved by the Institutional Animal Care and Use Committee at the University of South Florida, College of Medicine.

Induction of diabetes.

C57BL/6 mice (male, 13–15 wk old) were purchased from Jackson Laboratory. The mice were housed at 23 ± 1.0°C on a 12:12 h light-dark cycle with lights on at 6:00 AM and free access to standard chow and water. Diabetes was induced by an intravenous injection of alloxan (55 mg/kg in 100 µl saline) through the penile vein after overnight fasting. Blood glucose was measured twice a week starting 3 days after alloxan injection (Fig. 1). Mice with 300–500 mg/dl blood glucose levels were used in the present study. The mice with high blood glucose levels (>500 mg/dl) were treated with insulin (0.5 U/kg) to maintain the blood glucose at 300–500 mg/dl. The mice with an injection of 100 µl saline through the penile vein after overnight fasting served as nondiabetic animals. Measurement of blood glucose of the nondiabetic mice was the same as in the diabetic mice. All experiments were performed at 4 wk after alloxan or saline injection.

Fig. 1.

The change of blood glucose after alloxan injection.

Mean arterial pressure and renal hemodynamic responses to acute ANG II injection.

Mice were anesthetized with pentobarbital (50 mg/kg ip) and placed on a temperature-controlled operating table (Vestavia Scientific, Vestavia Hills, AL) to maintain body temperature at 37 ± 1.0°C. The carotid artery was catheterized for blood pressure measurement with a PowerLab (ADInstruments, Boulder, CO). Following an abdominal incision, the left renal artery was carefully separated from the vein, and a perivascular flow probe (Transonic Systems, Ithaca, NY) was placed around the left renal artery. The probe was then stabilized by a micromanipulator. Renal blood flow (RBF) measured with the flow probe was recorded via the PowerLab. After 30 min equilibration, mean arterial pressure (MAP) and RBF were measured for 5 min as a baseline. Then a bolus injection of 1 ng/kg ANG II (dissolved in 20 μl of 0.9% saline) was given through a penile vein (7). MAP and RBF were recorded for 15 min. Renal vascular resistance (RVR) was calculated as the renal artery pressure (same as MAP) divided by RBF.

MAP response to chronic ANG II infusion.

Telemetry transmitters (PA-C10, Data Sciences International) were implanted for measurement of MAP in conscious mice as we previously described (59, 102). Briefly, the mice were anesthetized with inhaled isoflurane (Butler chemicals) and placed on the temperature-controlled operating table. The left carotid artery was exposed by a small incision in the middle of the neck. The pressure catheter was implanted in the left carotid artery, and the telemetric device was placed subcutaneously in the right ventral flank of the mice. MAP was most stable from 1:00 to 5:00 PM during a 24 h period, so the MAP and heart rate were recorded for 10 s every 2 min for 4 h from 1:00 to 5:00 PM starting from 7 days after the transmitter implantation.

Five days after baseline MAP measurement, micro-osmotic pumps (model 1002, ALZET) were subcutaneously implanted as described previously (102). Briefly, micro-osmotic pumps were filled with ANG II (600 ng·kg⁻¹·min⁻¹) and incubated in sterile saline at 37°C overnight to reach steady state before implantation. Mice were anesthetized with isoflurane. A small incision was made in the skin between the scapulae, and a small pocket was formed by separating the subcutaneous connective tissues from the skin. The micro-osmotic pump was inserted into the pocket, and the wound was sutured. Fourteen days after implantation, the micro-osmotic pumps were removed from the animals and the new pumps filled with ANG II were subcutaneously implanted.

Isolation and microperfusion of Af-Art.

The isolation and microperfusion of the Af-Arts were the same as we described previously (58–60). Briefly, the mice without ANG II infusion were anesthetized with inhaled isoflurane, and the kidneys were removed and sliced. The kidney slices were placed in ice-cold Dulbecco's modified Eagle’s medium (DMEM). A single superficial intact glomerulus and its adherent Af-Art were microdissected under a stereomicroscope (SMZ1500; Nikon, Yuko, Japan). The microdissected sample was transferred to a temperature-regulated chamber mounted on an inverted microscope (Axiovert 100TV, Zeiss) together with DMEM. The glomerulus was held with micropipette, and Af-Art was cannulated and perfused with a set of micropipettes. The intraluminal pressure of the perfused Af-Art was maintained at 60 mmHg throughout the experiment. The chamber was perfused with DMEM with or without losartan (10⁻⁶ mol/l) at 1–1.5 ml/min at 37°C. After 30 min of an equilibration period, the dose-response curves of ANG II (10⁻¹² to 10⁻⁶ mol/l in DMEM, in random order) with or without losartan (10⁻⁶ mol/l) were obtained. Each concentration of ANG II was perfused for 5 min, and the maximum constrictive response of the Af-Art was measured; the bath was then switched to DMEM for 15 min before next dose of ANG II stimulation.

Real-time PCR.

The same method was used to isolate the Af-Art as we reported previously (59, 60, 102). Af-Arts were isolated from kidney slices of the mice without ANG II infusion in ice-cold DMEM under a stereomicroscope. A single Af-Art was transferred into RLT buffer (RNeasy Mini Kit; Qiagen, Venlo, Netherlands) for RNA extraction. To avoid RNA degradation, the time for dissection was limited to 30 min after kidney removal.

Total RNAs (20–30 ng/μl) were extracted from an Af-Art using RNase Mini Kit according to the manufacturer's instructions. After digestion with RNase-free DNase (Promega) to eliminate the genomic contamination, the cDNAs were synthesized with a reverse transcription system using oligo d(T) primer and used as templates. Quantitative PCR analysis was performed using iQ SYBR Green Supermix (Bio-Rad) and CFX96 Real-Time Detection System (Bio-Rad) according to the manufacturer's protocol. Reaction conditions for AT1 and AT2 receptors were 95°C for 1 min, followed by 40 cycles of 95°C for 15 s, then 60°C for 30 s; and for Mas receptor were 95°C for 3 min, 1 cycle; 95°C for 10 s, 56°C for 30 s, 50 cycles; 72°C for 10 s, 1 cycle (75). The reaction of each sample was performed in triplicate. Dissociation analysis was performed at the end of each PCR reaction to confirm the amplification specificity. After the PCR program, data were analyzed and quantified with the comparative C_t method (2^–ΔΔC_t) based on C_t values to calculate the relative mRNA expression level. The primer sequences and accession numbers are listed in Table 1. The expected size of PCR products of AT1, AT2, and Mas was 240, 230, and 175 bp, respectively (75, 98).

Table 1.

Mouse primer sequences and accession numbers

Gene Name	Sequences	Accession Number
AT1	forward: 5′-ATCGCTACCTGGCCATTGTC-3′	NM_177322.3
	reverse: 5′-GGAAGCCCAGGATGTTCTTG-3′
AT2	forward: 5′-TTACCAGCAGCCGTCCTTTT-3′	NM_007429.5
	reverse: 5′-GTCAGCCAAGGCCAGATTGA-3′
Mas	forward: 5′-AGGGTGACTGACTGAGTTTGG-3′	NM_008552
	reverse: 5′-GAAGGTAAGAGGACAGGAGC-3′
β-Actin	forward: 5′-GTCCCTCACCCTCCCAAAAG-3′	NM_007393.3
	reverse: 5′-GCTGCCTCAACACCTCAACCC-3′

Statistics.

The number of mice in each experiment was determined by power analysis based on P value = 0.05 and a power of 80% (16, 45). Data are presented as means ± SE. A Student’s t-test was used to determine statistical differences. An ANOVA with post hoc test was used for within-group and between-group measurements. A two-way ANOVA was used to compare dose response curves in isolated arterioles. The difference was considered to be significant for a P value < 0.05.

RESULTS

MAP and renal hemodynamic responses to an acute ANG II injection.

To determine whether the diabetic mice are more sensitive to acute ANG II stimulation, MAP and RBF were measured and compared between anesthetized nondiabetic and diabetic mice (Fig. 2A). Baseline MAP was 82.9 ± 1.1 and 83.7 ± 3.1 mmHg in diabetic and nondiabetic mice, respectively. MAP increased by 43.5 ± 2.6% to 119.1 ± 3.8 mmHg in diabetic mice following acute ANG II injection (P < 0.01 vs. baseline). However, MAP only increased by 26.8 ± 2.3% to 106.2 ± 3.5 mmHg in nondiabetic mice (Fig. 2, B and C) (P < 0.01 vs. baseline). Baseline RBF was significantly higher in diabetic mice (1.53 ± 0.18 ml/min) compared with nondiabetic mice (0.98 ± 0.12 ml/min) (P < 0.05 vs. nondiabetes). RBF decreased by 83.4 ± 5.4% to 0.25 ± 0.07 ml/min in diabetic mice (P < 0.01 vs. baseline) but only decreased by 47.1 ± 13.7% to 0.52 ± 0.14 ml/min in nondiabetic mice (P < 0.01 vs. baseline) following an acute ANG II injection (Fig. 2, D and E). Baseline RVR was significantly lower in diabetic mice compared with nondiabetic mice (Fig. 2F) (P < 0.05 vs. nondiabetes). RVR increased about eightfold in diabetic mice (P < 0.05 vs. baseline) and just increased ~1.5-fold in nondiabetic mice (Fig. 2G) (P < 0.05 vs. baseline). There was no functional response to an injection of 20 μl saline.

Fig. 2.

Responses of mean arterial pressure (MAP), renal blood flow (RBF), and renal vascular resistance (RVR) to acute ANG II injection. Representative image (A) demonstrates the change of MAP and RBF after acute ANG II injection. Acute ANG II injection induced a greater increase in MAP (B and C; *P < 0.01 vs baseline, #P < 0.05 vs nondiabetes) and a greater decrease in RBF (D and E; *P < 0.01 vs baseline, #P < 0.05 vs nondiabetes) in diabetic mice compared with nondiabetic mice. Calculated RVR was also greater in diabetic mice than in nondiabetic mice following ANG II infusion (F and G; *P < 0.05 vs. baseline, #P < 0.05 vs. nondiabetes) (diabetes, n = 10; nondiabetes, n = 7).

MAP response to chronic ANG II infusion.

To determine whether the sensitivity of blood pressure to chronic ANG II infusion is enhanced in diabetes, we measured MAP with telemetry in diabetic mice infused with a slow pressor dose of ANG II and compared with nondiabetic mice (Fig. 3A). Baseline MAP was 101.6 ± 3.4 mmHg in diabetic mice and 98.4 ± 2.2 mmHg in nondiabetic mice. MAP increased by 54.9 ± 6.1% to 157.4 ± 4.6 mmHg in diabetic mice following 4-wk ANG II infusion (P < 0.01 vs. baseline). However, MAP only increased by 32.8 ± 4.4% to 130.7 ± 3.6 mmHg in nondiabetic mice (Fig. 3, B and C) (P < 0.01 vs. baseline). There were no significant differences in heart rate between diabetes and nondiabetes (Fig. 3D).

Fig. 3.

Changes in MAP in response to chronic ANG II infusion. Chronic ANG II infusion caused a greater increase in MAP in diabetic mice compared with nondiabetic mice. (n = 6) *P < 0.01 vs baseline; #P < 0.01 vs nondiabetes.

Diabetes enhances the constrictive effect of ANG II on Af-Art.

To determine whether ANG II-induced constriction of the Af-Art is enhanced in diabetes, we measured the dose-response curves for ANG II (10⁻¹² to 10⁻⁶ mol/l) in isolated perfused Af-Art in diabetic mice and compared them with those of nondiabetic mice (Fig. 4A). In diabetic mice, the basal diameter of the Af-Art was 12.91 ± 0.3 μm. ANG II (10⁻¹² to 10⁻⁶ mol/l) reduced the Af-Art diameter in a dose-dependent manner. The diameter was reduced to 56.3 ± 3.2% of baseline at 10⁻⁹ mol/l ANG II (P < 0.01 vs. baseline) and 34.1 ± 4.3% of baseline at 10⁻⁶ mol/l ANG II (P < 0.01 vs. baseline). In nondiabetic mice, the Af-Art diameter was 11.76 ± 0.9 μm at baseline and reduced to 83.1 ± 6.6% of baseline at 10⁻⁹ mol/l of ANG II (P < 0.05 vs. baseline) and to 52.1 ± 5.3% of baseline at 10⁻⁶ mol/l of ANG II (P < 0.01 vs. baseline). ANG II-induced contraction was significantly enhanced in diabetic mice (Fig. 4, B and C) (P < 0.05 vs. nondiabetes).

Fig. 4.

Dose-response curve of the afferent arteriole (Af-Art) to ANG II. Representative images (A) demonstrated Af-Art constriction induced by ANG II in nondiabetic and diabetic mice. ANG II-induced contraction was significantly enhanced in diabetic mice (B and C; *P < 0.05 vs. nondiabetes). (diabetes: n = 11, nondiabetes: n = 8). AT1 receptor antagonist losartan blocked ANG II-induced (10⁻¹² to 10⁻⁶ mol/l) vasoconstriction of the Af-Art in both diabetic and nondiabetic mice. (D and E) (diabetes, n = 7; nondiabetes, n = 6).

Losartan blocked ANG II induced vasoconstriction.

To determine whether ANG II constricts Af-Art via activation of AT1 receptors, the dose-response curve for ANG II (10⁻¹² to 10⁻⁶ mol/l) plus losartan (10⁻⁶ mol/l) was measured and compared between diabetic and nondiabetic mice. ANG II (10⁻¹² to 10⁻⁶ mol/l) did not reduce the Af-Art diameter in the presence of losartan in diabetic and nondiabetic mice (Fig. 4, D and E).

mRNA levels of RAS receptors.

To determine whether diabetes alters the expression levels of angiotensin receptors, we dissected an Af-Art from nondiabetic and diabetic mice (8 wk after alloxan injection) respectively and measured the mRNA expressions of AT1, AT2 and Mas receptors by real-time PCR. The AT1 receptor mRNA level in the Af-Art was 9.4 ± 0.5 times higher in diabetic mice than that in nondiabetic mice (P < 0.01 vs. nondiabetes) (Fig. 5A). The AT2 and Mas receptor mRNA levels were similar in diabetic and nondiabetic mice (Fig. 5, B and C).

Fig. 5.

mRNA levels of AT1 and AT2 receptors in the Af-Art. AT1 receptor mRNA level was significantly higher in diabetic mice than that in nondiabetic mice in the isolated Af-Arts (A; *P < 0.01 vs. nondiabetes). AT2 and Mas receptors mRNA level in diabetic mice was similar as that in nondiabetic mice in the Af-Arts (B and C) (diabetes, n = 10; nondiabetes, n = 10).

DISCUSSION

In the present study, we demonstrated that the responses of MAP and RBF to both acute and chronic ANG II infusion were enhanced in alloxan-induced diabetic C57BL/6 mice. The constrictive response of the Af-Art to ANG II was exaggerated in diabetic mice associated with upregulated expression of AT1 receptor. AT1 receptor antagonist losartan blocked ANG II induced vasoconstriction in both nondiabetic and diabetic mice.

Diabetic patients have a higher prevalence of hypertension than nondiabetic subjects, but the reason has not been completely understood. RAS inhibition showed a distinctive benefit on cardiovascular outcomes for diabetic patients with hypertension (2, 78), suggesting a critical role of the RAS in the development of hypertension in diabetes (1, 71). Even though the plasma renin activity was suppressed (18, 27, 77) or normal (11, 61) in diabetes, the response of blood pressure to acute ANG II infusion was more sensitive in diabetic patients compared with nondiabetic subjects (10, 24, 100, 101). Similar findings have been reported in diabetic animal models. Plasma renin concentration (39, 94), renin activity (39, 47, 76), and ANG II activity (47) were decreased in diabetic models, but the response of blood pressure to chronic administration of AT1 receptor antagonist losartan was enhanced (85). In the present study, we compared MAP in diabetic and nondiabetic mice in response to either bolus intravenous injection or chronic infusion of ANG II. The higher MAP in the diabetic group indicates that the responsiveness of blood pressure to ANG II is exaggerated in diabetic mice.

Renal hemodynamics play an important role in control of salt-water balance and blood pressure, and hypertension is usually associated with decreased RBF and increased RVR (70, 74, 83). Type 1 diabetes is characterized by impairment in autoregulation, reduction in RVR, and elevation in RBF, particularly during the early stage of diabetes (8, 68, 92). In the present study, we demonstrated that the diabetic mice exhibited normal blood pressure but increased RBF compared with nondiabetics, suggesting the impaired autoregulation and decreased RVR in diabetes. Previous studies have demonstrated that both acute and chronic ANG II infusion intensify blood pressure along with a marked increase of RVR in nondiabetics (26, 35, 96). However, the renal hemodynamic responses to ANG II in diabetes are not known yet. In the present study, we compared RBF and calculated RVR between diabetic and nondiabetic mice in response to bolus intravenous injection of ANG II. We found that the renal hemodynamic response to acute ANG II infusion was greater in diabetic mice compared with nondiabetic mice.

As the major resistance vessels in the kidney, Af-Arts account for >50% of the RVR (6, 13, 15). To investigate the vascular response to ANG II in diabetes, we compared the ANG II dose-response curves of the Af-Arts from diabetic and nondiabetic mice. We found that the vasoconstriction of renal Af-Art in response to ANG II was significantly exaggerated in diabetic mice. Consistent with our finding, enhancement in ANG II-dependent vasoconstriction has been found in several different kinds of vessels, including mesenteric, carotid, and renal arteries from diabetic rats, indicating that increased responsiveness to ANG II may be a general characteristic of the vasculatures in diabetes (9).

Effects of ANG II on the Af-Arts are primarily mediated by two types of ANG II receptors, AT1 and AT2 receptors (31, 38, 43). Activation of the AT1 receptor in vascular smooth muscle cells stimulates Ca²⁺ influx and subsequently induces vasoconstriction (42, 56). On the contrary, AT2 receptor mediates the endothelium-dependent vasodilatation via stimulation of nitric oxide (NO) and bradykinin (5, 88, 89). The renal expression of angiotensin receptors in diabetes has been investigated by several laboratories. The renal AT1 receptor protein level in diabetic rats was reported to be higher than in nondiabetic rats (37). However, several other studies report that the kidney AT1 receptor was downregulated in diabetic animal models and diabetic patients (14, 65, 104). Also, a lower expression level of the AT1 receptor was found in hypertensive animals with diabetes (12, 14, 104). In addition, Wagner et al. (97) demonstrated that the mRNA level of the AT1 receptor was significantly lower in patients with Type II diabetes than nondiabetics in kidney biopsy samples. The reasons for the inconsistent observations in AT1 expression levels in diabetes are not clear but might be due to the different regions of the kidney that were examined. Regarding the AT2 receptor, a downregulation of expression in the kidney was demonstrated in both early streptozotocin-induced diabetic Sprague-Dawley rats (99) and spontaneously hypertensive rats with long-term diabetes (12). Moreover, the localization of ANG II receptors is widely distributed in the kidney. Both immunohistochemical and ANG II binding studies demonstrated that AT1 receptor was abundant in the cortical vasculature and S3 segment of the proximal tubules in the outer medulla, with lesser expression in the thick ascending limb and collecting ducts (63, 64, 67). The major distribution of AT2 receptor in the kidney was indicated in interlobular arteries, Af-Arts, glomeruli, proximal tubules, and collecting ducts (62, 87, 99). Thus, the expression levels of ANG II receptors in the kidney homogenate may not reflect their specific levels in Af-Art. To determine the expression of ANG II receptors exclusively in Af-Art, we isolated Af-Art and then measured the mRNA level of AT1 and AT2 receptors. We found that AT1 receptor expression was over 10-fold higher in mice at 8 wk of diabetes than in nondiabetic mice, whereas the level of AT2 receptor was similar. This finding is consistent with the study by Sodhi et al. (90) that found the medium with high glucose upregulated AT1 receptor expression in cultured vascular smooth muscle cells. However, our results are not inconsistent with the previous report that both AT1-A and AT1-B receptor protein levels in renal arterioles, measured by immunohistochemistry, were lower in STZ-induced diabetic Wistar rats (32, 81). The difference in blood pressure between diabetic and nondiabetic mice was not significant until 2 wk after ANG II infusion or 6 wk following alloxan injection. The reasons are not clear and may be due to the compensatory mechanisms or low expression level of AT1 in the Af-Art within the first few weeks of diabetes.

Besides the direct vascular reactions via AT1 and AT2 receptors, ANG II is converted by angiotensin converting enzyme-2 (ACE-2) into angiotensin 1–7 (ANG 1–7), which binds to G protein-coupled receptor Mas counteracting the AT1 receptor-mediated vasoconstriction. Previous studies demonstrated that the expression of renal ACE-2 and Mas receptor was decreased in 20 wk Akita mice (86). As the Mas receptor has an extensive distribution in the kidney, including the proximal tubule, thick ascending limb, and collecting duct, to determine the expression of Mas receptor exclusively in Af-Art, we isolated Af-Art and measured the mRNA level of Mas receptors. Inconsistent with the expression of Mas receptors in the kidney homogenate, our results showed that there is no significant change of Mas receptor expression in the Af-Arts between diabetic and nondiabetic mice, which suggests that the ACE-2/ANG 1–7/Mas pathway might not play a significant role in the enhancement of ANG II-induced Af-Art constriction in diabetic mice.

NO is an important modulator that negatively regulates the AT1 receptor-mediated vasoconstriction. NO in renal Af-Art is primarily generated in endothelial cells via endothelial nitric oxide synthase (NOS3). Previous evidence demonstrated that the expression of NOS3 in the endothelium of Af-Arts was markedly increased in alloxan-induced diabetic C57BL/6 mice (103), which suggests that NOS3-dependent NO generation should counteract the vasoconstriction of renal Af-Art in response to ANG II in diabetic mice. Both type 1 and type 2 cyclooxygenase (COX) are identified in renal Af-arts (22, 36). Prostaglandins (PGs) generated by the COX have been implicated in modulation of ANG II-induced Af-Art vasoconstriction (30, 80). Inhibition or knockout of COX2 augments the pressor effect of ANG II (48, 79). Vasodilatory PGs, such as prostacyclin and prostaglandin E2 (PGE2), are involved in the hyperfiltration in the early stage of diabetes (19, 40, 44). Even though the changes in COX expression of Af-Arts in diabetes are not clear, several studies have demonstrated that renal expression of COX2 (49, 54, 66) or PGE2 (17, 41) is upregulated in diabetes, which suggests that COX-derived PGs are unlikely to promote the ANG II-induced vasoconstriction of renal Af-Art in diabetic mice.

The synergistic interaction of contractile response between ANG II and adenosine on renal Af-Art has been recognized (15, 55, 73). In the presence of adenosine, the ANG II-induced vasoconstriction of Af-Art was significantly enhanced (55, 73). However, compared with wild-type mice, adenosine A1-receptor knockout mice exhibited similar renal hemodynamic changes in alloxan-induced diabetes (84), suggesting that adenosine is not the major cause of the enhanced renal hemodynamic responses or vasoconstriction of renal Af-Art to ANG II in diabetes.

Taken together, these findings indicate that exaggerated Af-Art vasoconstrictive response to ANG II, mediated by the increased expression of AT1 receptor, may contribute to the enhanced blood pressure and renal hemodynamic responses to ANG II in diabetes. Therefore, the current study reveals a mechanism that may contribute to the development of hypertension in diabetes.

Perspectives and Significance

Diabetic subjects have a higher prevalence of hypertension, but the mechanisms have not been fully elucidated. We demonstrated in the present study that diabetes exaggerates the constrictive responsiveness of the Af-Art to ANG II, which was mediated by the upregulation in the expression of AT1 receptor. This mechanism may promote the enhanced response of blood pressure and renal hemodynamic to ANG II in diabetes. Plasma renin-angiotensin II level is usually suppressed in early diabetes (3, 46), which may permit a low vascular tone of the Af-Art and a high glomerular hyperfiltration in early diabetes. On the other hand, mild or moderate elevation of circulating and/or local renin-angiotensin II that may not increase blood pressure in nondiabetic patients could induce hypertension in diabetes, which may be one of the mechanisms for the high prevalence of hypertension in diabetes. The findings of the present study reveal the significance of RAS as a potential mechanism for the hypertension in diabetes and justify the essential role of the RAS inhibition for the diabetic patients with hypertension.

GRANTS

This work was supported by AHA-15PRE2571306 (to J. Zhang) and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-099276 and DK-098582 (to R. Liu).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.Z., J.W., and R.L. conceived and designed research; J.Z., J.S., J.W., and S.J. performed experiments; J.Z., H.Y.Q., J.S., J.W., S.J., and R.L. analyzed data; J.Z., H.Y.Q., and J.S. prepared figures; J.Z. and J.S. drafted manuscript; J.Z., J.S., J.W., S.J., Lei Wang, Liqing Wang, J.B., and R.L. approved final version of manuscript; H.Y.Q., J.S., J.W., S.J., Lei Wang, Liqing Wang, J.B., and R.L. edited and revised manuscript.